The aim of this study was to evaluate the effect of osteopontin (OPN), an ubiquitous acid glycoprotein, on in vitro sperm capacitation and on in vitro embryo production (IVEP) efficiency in buffalo. In experiment 1, after swim-up separation the sperm were incubated in Tyrode albumin lactate pyruvate medium in the absence of capacitating agents (control), with the standard concentration of heparin (0.01 mM) and three different concentrations of OPN (0.1, 1, and 10 mcg/mL), both in the presence and absence of heparin, for 2 and 4 hours. Capacitation was assessed indirectly by estimating the percentage of acrosome-reacted sperm after incubation with lysophosphatidylcholine. In order to determine the effect of OPN, in the presence of heparin, on fertilization (Experiment 2) and in vitro embryo development (experiment 3), in vitro-matured buffalo oocytes were fertilized in the presence of 0, 0.1, 1, and 10 mcg/mL of OPN. After IVF, the presumptive zygotes were dezonated, fixed, stained, and then evaluated microscopically. At Days 5 and 7 of culture, the cleavage and blastocyst rates were evaluated, respectively. Two hours of treatment with OPN at the two higher concentrations (1 and 10 mcg/mL) promoted in vitro capacitation of buffalo sperm (experiment 1). A synergic action of OPN with heparin was also done for all OPN concentrations tested. At 4 hours incubation, all treatments, including heparin (20.4%), improved (P < 0.01) capacitation compared with the control (16.2%). Interestingly, the best results were reported in all groups treated with OPN + heparin (40.8%, 38.6%, and 33.8%, respectively; P < 0.01). The addition of OPN to the IVF medium had a positive influence on total penetration, synchronous pronuclei formation (experiment 2), and IVEP efficiency (experiment 3). In particular, the two lower concentrations of OPN (0.1 and 1 mcg/mL), compared with the control, gave higher synchronous pronuclei formation (73.5%, 75.0%, and 46.5%, respectively; P < 0.01) and cleavage rates (70.3%, 71.6%, and 59.3%, respectively; P < 0.01). Interestingly, the treatments also improved blastocyst yields (29.3%, 30.3%, and 19.4%, respectively; P < 0.01). In conclusion, these results indicate that adding OPN to the IVF system improves IVEP efficiency by enhancing in vitro sperm capacitation and blastocyst yields in buffalo.