Mammalian spermatozoa must complete an acrosome reaction prior to fertilizing an oocyte. The acrosome reaction is a unique exocytotic event involving a series of prolonged membrane fusions that ultimately result in the production of membrane vesicles and release of the acrosomal contents. This event requires the concerted action of a large number of fusion-competent signalling and scaffolding proteins. Recently we have shown that two members of the dynamin family of large GTPases localize to the developing acrosome of maturing mouse germ cells and participate in the regulation of acrosomal exocytosis. Herein, we have extended our analysis of these dynamin family members to human spermatozoa to determine if they play an analogous role in promoting the acrosome reaction in our own species Through the use of Western blotting and immunocytochemistry we were able to confirm the presence of both dynamin 1 and dynamin 2 in human spermatozoa, and demonstrate that both proteins localise to the outer acrosomal region of the sperm head, an ideal position from which to regulate the exocytotic release of the acrosomal contents. Consistent with this proposed role, we were also able to demonstrate that pharmacological inhibition of dynamin activity led to a significant reduction in the ability of human spermatozoa to acrosome react. Furthermore, we have identified reduced levels of dynamin expression in spermatozoa recovered from infertile patients with known defects in oocyte penetration. Collectively these data support an important and conserved role for dynamin in the regulation of mammalian sperm function.