The two subspecies of the North American bison, plains (Bison bison bison) and wood (Bison bison athabascae) bison, are seasonal breeders. The objective of this study was to conduct a preliminary investigation into the effects of season on semen. To test the hypothesis that there are seasonal effects on seminal plasma, protein profiles of seminal plasma from plains and wood bison (n = 2 of each subspecies) were compared between breeding and nonbreeding seasons. Using two-dimensional gel electrophoresis of seminal plasma proteins, 54 of 170 spots (plains bison) and 19 of 153 spots (wood bison) had differential expression (≥2-fold; P < 0.01) between seasons. Based on immunoblotting, BSP5 and TIMP-2 (two fertility-associated proteins in cattle) were higher during the breeding vs. nonbreeding season. Furthermore, epididymal sperm incubated with seminal plasma from the nonbreeding season had lower postthaw progressive motility (17.33 ± 7.47 vs. 22.09 ± 6.67%; mean ± SD) and an increased ability to undergo a lysophosphatidylcholine-induced acrosome reaction (77.83 ± 8.47 vs. 52.67 ± 7.76%; mean ± SD) as compared to epididymal sperm incubated with seminal plasma from the breeding season. In a heterologous in vitro fertilization system (using bovine oocytes), cleavage rate was higher for sperm exposed to seminal plasma from the breeding vs. the nonbreeding season (75.35 ± 16.55 vs. 33.63 ± 12.44%; mean ± SD). This study suggested that differential expression of seminal plasma characteristics modulating sperm function is one of the mechanisms by which reproductive seasonality affects sperm function in the North American bison.