Phospholipase A2 (PLA₂) plays a major role during acrosomal exocytosis (AE) in mammalian spermatozoa, but the identity of PLA₂subtypes present in spermatozoa remains elusive. This study explored whether secretory PLA₂ Group IID (sPLA₂‐IID) isoform is present in human spermatozoa and whether it is involved in AE. Localization and expression of sPLA₂‐IID in human spermatozoa were explored by immunofluorescence staining and Western blot analysis. Occurrence of AE was evaluated by triple staining, and arachidonic acid (AA) levels were quantified by gas chromatography—mass spectrometry. Sperm motion parameters and hyperactivation were analyzed by computer‐assisted sperm analysis. sPLA₂‐IID was localized in the postacrosomal region of the head and the midpiece of tail in human sperm. A 16‐kd protein band was detected by Western blotting in sperm extracts. Progesterone‐induced AE was significantly inhibited in a concentration‐dependent manner using a sPLA₂‐IID neutralizing antibody. The increase in AA levels seen during progesterone‐stimulated exocytosis was significantly abrogated by the antibody. The sPLA₂‐IID antibody significantly inhibited hyperactivation, sperm curvilinear velocity, and amplitude of lateral head displacement, but it did not affect the proportion of motile sperm. In conclusion, sPLA₂‐IID is present at the head and midpiece in the human sperm, and activation of such sPLA₂‐IID seems to be involved in AE. Therefore, sPLA₂‐IID isoform plays a functional role during the AE in human sperm.