Sperm must undergo a process termed capacitation in the female reproductive tract before they are capable of fertilization. Previous studies strongly suggest that capacitation of sperm occurs in the oviduct. The secreted extracellular matrix phosphoprotein osteopontin (OPN) has been positively correlated to fertility in Holstein bull seminal plasma and identified in the bovine oviduct, where we hypothesized it plays a role in sperm capacitation. To determine the effect of OPN on sperm capacitation, sperm were incubated in OPN purified from bovine milk in concentrations ranging from 1 to 20 µg/ml and subjected to dual fluorescence acrosomal and viability staining. Sperm were also incubated in casein (CSN; 1 to 20 µg/ml) and isthmic non-luteal oviductal fluid (INLODF), ampullary non-luteal oviductal fluid (ANLODF) and skim milk. Flow cytometry analysis showed that OPN induced capacitation at low concentration; the largest percentage of capacitated sperm followed after incubation in INLODF. Sperm intracellular calcium levels, an indicator of sperm capacitation, were increased by OPN and INLODF, and OPN had an overall positive effect on sperm viability. There was, however, no effect of any treatment on sperm mitochondrial activity. Experiments utilizing biotinylated OPN and CSN demonstrated that CSN, but not OPN, bound to sperm. The results presented here offer the first direct evidence of the capacitating effect of OPN on bovine sperm and provide confirmation of the increasing number of roles this fascinating protein plays in reproductive biology.