Jake Blues is released from prison after serving three years, and he is picked up by his brother Elwood in his Bluesmobile, a battered former police car. Elwood demonstrates its capabilities by jumping an open drawbridge. The pair visit the Roman Catholic orphanage where they were raised, and they learn from Sister Mary Stigmata that it will be closed unless $5,000 in property taxes is collected. During a sermon by the Reverend Cleophus James at the Triple Rock Baptist church, Jake has an epiphany: they can re-form their band, the Blues Brothers, which disbanded while Jake was in prison, and raise the money to save the orphanage.
That night, state troopers attempt to arrest Elwood for driving with a suspended license due to his 116 parking tickets and 56 moving violations. After a high-speed chase through the Dixie Square Mall, the brothers escape. As they walk into the flophouse where Elwood lives, an unknown woman fires a multiple barrelled rocket launcher at Jake, destroying the building's entrance, but somehow leaving the brothers unharmed. The next morning, during a police raid, the same woman detonates a bomb that demolishes the building but, miraculously, again leaves Jake and Elwood unharmed, although a squad of police attempting to apprehend them are killed.
Jake and Elwood begin tracking down members of the band. Five of them are playing a deserted Holiday Inn lounge and quickly agree to rejoin. Another turns them down protesting that he's the maître d' at an expensive and elegant restaurant, but the brothers behave unbecomingly until he relents. On their way to meet the final two band members, the brothers find the road through Jackson Park blocked by an American Nazi Party demonstration on a bridge; Elwood runs them off the bridge into the East Lagoon, and the commander orders a subordinate to write down their vehicle's license plate. They lastly invite the final two band members, who now run a soul food restaurant; the brothers blackmail one with threats against their children, and they rejoin the band against the advice of one's wife. The reunited group obtain instruments and equipment from Ray's Music Exchange in Calumet City, and Ray, as usual, takes an IOU.
As Jake attempts to book a gig, the mysterious woman uses an M9-7 Flamethrower to blow up the phone booth he is using, which is situated next to a fuel tank. For a third time, he is miraculously unhurt. The band stumbles into a gig at Bob's Country Bunker, a local honky-tonk. They win over the rowdy crowd, but they run up a bar tab higher than their pay and infuriate the country band that was actually booked for the gig, the Good Ol' Boys.
Realizing that they need one big show to raise the necessary money, the brothers persuade their old agent to book the Palace Hotel Ballroom, north of Chicago. They mount a loudspeaker atop the Bluesmobile and drive the Chicago area promoting the concert. However, they inadvertently alert the police, the Nazis, and the Good Ol' Boys of their whereabouts. The ballroom is packed with blues fans, law enforcement, and the Good Ol' Boys. Jake and Elwood perform two songs, then sneak offstage, as the tax deadline is rapidly approaching. A record company executive offers them a $10,000 cash advance on a recording contract—more than enough to pay off the orphanage's taxes and Ray's IOU—and then shows the brothers how to slip out of the building unnoticed. As they make their escape via a service tunnel, they are confronted by the mystery woman: Jake's vengeful ex-fiancée. After her volley of M16 rifle bullets leaves them miraculously unharmed, Jake offers a series of ridiculous excuses that she accepts, allowing the brothers to escape to the Bluesmobile.
Jake and Elwood race back toward Chicago with dozens of state/local police and the Good Ol' Boys in pursuit. They eventually elude them all with a series of improbable maneuvers, including a miraculous gravity-defying escape from the Illinois Nazis. At the Richard J. Daley Center, they rush inside the adjacent Chicago City Hall building, soon followed by hundreds of police, state troopers, SWAT teams, firefighters, Illinois National Guardsmen, and the Military Police. Finding the office of the Cook County Assessor, the brothers pay the tax bill. Just as their receipt is stamped, they are arrested by the mob of law officers. In prison, the band plays "Jailhouse Rock" for the inmates.
John Belushi as "Joliet" Jake Blues, a former blues singer, paroled from prison after three years.
Dan Aykroyd as Elwood Blues, Jake's blood brother, also a former blues singer.
James Brown as Reverend Cleophus James, a reverend of the Triple Rock Baptist Church. His musical sermon "The Old Landmark" causes Jake to have an epiphany.
Cab Calloway as Curtis, an old friend of the brothers', who suggests they listen to James, and helps them advertise the show and performs "Minnie the Moocher" for the audience.
Ray Charles as Ray, a blind music instruments dealer, who performs "Shake a Tail Feather" to demonstrate their effectiveness.
Aretha Franklin as Mrs. Murphy, Matt Murphy's wife, who owns a soul food restaurant with him. She performs "Think" to persuade him not to join the band.
Steve "the Colonel" Cropper – lead guitar
Donald "Duck" Dunn – bass guitar
Murphy Dunne ("Murph") – keyboards
Willie "Too Big" Hall – drums
Tom "Bones" Malone – trombone
"Blue Lou" Marini – saxophone
Matt "Guitar" Murphy – lead guitar
"Mr. Fabulous" Alan Rubin – trumpet
Carrie Fisher as the Mystery Woman, Jake's former fiancée; she tries to kill him for leaving her at the altar.
Henry Gibson as the Head Nazi, the head of a division of the American Socialist White People's Party
John Candy as Burton Mercer, Jake's parole officer assisting the police in their hunt for the Blues Brothers.
John Lee Hooker as Street Slim, a man singing "Boom Boom" together with a small band on Maxwell Street.
Kathleen Freeman as Sister Mary Stigmata, a nun leading the orphanage where the brothers grew up.
Steve Lawrence as Maury Sline, the agent who organized and booked many of the Blues Brothers' performances before Jake was sent to jail.
Twiggy as the Chic Lady, a woman who flirts with Elwood at the gas station.
Frank Oz as a corrections officer
Jeff Morris as Bob, the owner of Bob's Country Bunker.
Charles Napier as Tucker McElroy, lead singer and Winnebago driver of The Good Old Boys.
Steven Spielberg as the Cook County Assessor
Steven Williams as Trooper Mount
Armand Cerami as Trooper Daniel
Joe Walsh as Prison Inmate
Paul Reubens as Waiter
Chaka Khan as Choir Soloist