The oviduct is a crucial organ for fertilization and has been demonstrated to perform a variety of interactions with spermatozoa ranging from sperm storage, to stabilizing sperm membranes and reducing free radicals. The oviduct is separated into 2 anatomically and physiologically distinct regions: the isthmus, in which sperm are stored, and the ampulla where fertilization occurs. We aimed to investigate whether proteins derived from different regions of the bovine oviduct had beneficial effects on bovine sperm membrane integrity, osmotic resistance, and motility following cryopreservation. The extent to which sperm motility could be activated by bicarbonate was demonstrated and used as a novel approach to postthaw sperm assessment. While oviductal proteins did not increase the degree of postthaw sperm viability, spermatozoa exposed to the isthmic proteins before freezing showed higher osmotic resistance after thawing. The presence of bicarbonate increased the proportion of spermatozoa with high curvilinear (VCL) and straight line velocity (VSL) in all treatment groups. After thawing, spermatozoa exposed to isthmic proteins had higher VCL and VSL than spermatozoa exposed to the ampullar proteins. We conclude that proteins derived from the isthmus can stabilize and protect spermatozoa during cryopreservation.