Objective The maturation of mammalian epididymal sperm and the acquisition and maintenance of motility are prerequisites for normal sperm function and complete fertilization; however, the mechanism that regulates this process is still unclear. SRC kinase is involved in the regulation of mouse sperm capacitation, and the serine / threonine phosphatase PP1γ2 / PP2A is the key enzyme that regulates epididymal sperm maturation and motility. However, it is unclear whether these molecules interact with each other and whether this interaction regulates sperm motility ( including motility acquisition). In this study, we investigated the role of SRC kinase and phosphatase PP1γ2 / PP2A in Kunming mouse sperm and their regulation of sperm functions. Methods Using western blot, enzyme activity assay and co- immunoprecipitation assay, we examined threonine phosphorylation level as well as enzyme activity of SRC kinase and phosphatase PP1γ2 / PP2A in Kunming mouse sperm from caput and cauda epididymis. In addition, we investigated the interaction of SRC kinase and phosphatase PP1γ2 / PP2A.We also investigated the effects of a SRC inhibitor (SU6656) and an activator (sc-3052) on phosphatase activity and motility of sperm from cauda and caput epididymis. The phosphorylation level of threonine in sperm of cauda epididymis was higher than that in caput epididymis. SRC kinase activity in sperm from caput epididymis was lower than that in epididymal cauda sperm. The phosphatase activity of sperm from caput epididymis was significantly higher than that from cauda epididymis (P< 0. 05). SRC kinase in mouse epididymal sperm modulates phosphatase PP1γ2 or PP2A, which in turn influences sperm motility. Results In epididymal cauda sperm, where SRC is more active, when SRC activity is inhibited by SU6656, the activity of phosphatase PP1γ2 / PP2A is increased, while sperm motility is decreased. In epididymal caput sperm where SRC is less active, when SRC activity is enhanced by sc- 3052, the PP1γ2 / PP2A phosphatase activity is reduced, while sperm motility is increased. Conclusions The activity of SRC and phosphatase PP1γ2 / PP2A in caput sperm from mouse epididymis is significantly different from that in cauda sperm. SRC kinase interacts with phosphatase PP1γ2 or PP2A in mouse sperm. SRC kinase modulates sperm motility (including motility acquisition) by inhibiting the activity of phosphatase PP1γ2 / PP2A.