AbstractDifferent mammalian secreted phospholipases A2 (sPLA2s) are expressed in male reproductive organs and/or in sperm cells but their cellular functions are still not fully characterized. Because several reports indicate a link between cellular lipids and sperm motility, we have investigated the effect of mouse group IIA, IID, IIE, V, and X sPLA2s on sperm motility. Among these enzymes, only mouse group X sPLA2 (mGX sPLA2) acts as a potent inhibitor of sperm motility that decreases track speed (VCL) and lateral displacement of the head (ALH) of both noncapacitated and capacitated sperm. The inhibitory effect of mGX sPLA2 is dependent on its enzymatic activity because (i) both the proenzyme form of mGX sPLA2 (pro‐mGX) and the H48Q mutant of mGX sPLA2 have very weak enzymatic activity and are unable to modulate sperm motility and (ii) LY329722, a specific inhibitor of sPLA2s, blocks the inhibitory effect of mGX sPLA2. Moreover, mGX sPLA2 exerts a gradual potency on sperm subpopulations with different velocities, an effect which may be linked to the heterogeneity of lipid composition in these sperm subpopulations. Finally, we found that endogenous mGX sPLA2 released during spontaneous acrosome reaction modulates sperm motility of capacitated sperm. Together, our results suggest a new role of sPLA2 in sperm physiology where the sPLA2 selects a sperm subpopulation for fertilization based on its effect on sperm motility. J. Cell. Physiol. 226: 2601–2609, 2011. © 2010 Wiley‐Liss, Inc.