Bison face a significant threat from humans hunting their populations through selective harvesting or trophy hunting. Although the impact of hunting on the demography of bison is thoroughly documented, the effects on the behavior of surviving bison are not understood to the same degree. In North American plains bison, Bison bison, ‘bellows’ are known as low, guttural vocalizations made by bulls during the breeding season in male-male contests. One critical area of bison behavior that hunting could affect is formant frequencies, which are peaks with a higher degree of energy. Vocal tract dimensions determine formant frequencies, and longer vocal tracts produce lower frequencies. They can be reliable indicators of body size and other important traits for mating selection. Additionally, this study investigated if there was a change in the bellow duration due to human hunting. We analyzed samples of bison recordings from nine locations in South Dakota and Montana. We found that the formant frequency values of the hunted population were significantly higher than those of the non-hunted population. Additionally, bellow durations were shorter among hunted populations. The results indicate that hunting may cause smaller body sizes in males, which could negatively affect the demographics of bison populations. There are also greater implications for adaptive evolution, and perhaps adjustments needed to be made to hunting practices to ensure bison can engage in their natural mating behaviors.