In almost all animal species, sperm acrosome reaction (AR) is a crucial step for fertilization. The step is a Ca2+-dependent secretory event that must be completed before fertilization. Many researchers have reported that several chemicals (such as ionomycin, thapsigargin and caffeine) artificially induce this step by increasing [Ca2+]i. However, little information has been known on events that occur following Ca2+ induced initiation of the sperm AR. We show here for the first time that phosphorylation of the mitogen-activated protein kinase (MAPK) pathway is required for the AR in miniature pig sperm. Following caffeine treatment artificially inducing the AR in miniature pig sperm, Raf was phosphorylated and then MAP kinase kinase (MEK) and extracellular-signal regulated kinase1 (ERK1) were also phosphorylated in a time-dependent manner. However, the total ERK1 level did not change during the culture. Pre-treatment of sperm with U0126, a MEK inhibitor, significantly suppressed both the AR and phosphorylation of MEK/ERK1 in a dose-dependent manner. Additionally, pre-incubation of the sperm with seminal vesicle (SV) fluid, which is known to contain a decapacitation factor, suppressed both the AR and MEK/ERK1 phosphorylation. These results suggest that phosphorylation of MAPK pathway plays an important role in the AR in miniature pig sperm. Moreover, the SV fluid may have an inhibitory effect on the AR via the suppression of the MAPK pathway.