The porcine zona pellucida (ZP) undergoes biochemical changes during the final phase of maturation prior to fertilization. The present study was conducted to elucidate whether the acidification of ZP glycoproteins during porcine oocyte maturation influences sperm-ZP interactions. Two-dimensional gel electrophoresis clearly demonstrated that ZP acidification occurred in accordance with the sialylation and sulfation of ZP glycoproteins in oocytes matured for 44 h. The increases in the incidences of sperm penetration and polyspermy with the progress of the IVM culture period were significantly suppressed by ZP desialylation on treatment with neuraminidase as a consequence of reductions in the number of sperm bound to ZPs and the acrosome reaction (AR) in ZP-bound sperm (P<0.05). In contrast, the blocking of ZP sulfation by NaClO₃ treatment during IVM markedly reduced the incidence of polyspermy with no inhibitory effect on penetration, but the number of sperm bound to ZPs and the rate of AR-inducing sperm were decreased to the same level as in desialylated oocytes. The results indicate that ZP sulfation influences sperm-ZP interactions in a ZP sialylation-independent manner. Moreover, sialylation and sulfation were not associated with a protective proteolytic modification of the ZP matrix before fertilization. These findings suggest that ZP acidification elicited by the sialylation and sulfation of ZP glycoproteins during oocyte maturation contributes to the porcine ZP acquiring the capacity to accept sperm.