The gene encoding the protein known as “corticosteroids and regional expression 16” (CR16) has been shown to be regulated by glucocorticoids. CR16 is a member of the Wiskott–Aldrich syndrome protein (WASP)‐interacting protein (WIP) family. It binds to the neural WASP (N‐WASP), which activates the Arp2/3 complex to induce actin polymerization. CR16 is highly expressed in the testes, particularly in the Sertoli cells, which harbor sperm progenitors and play an important role in spermatogenesis. We found male‐specific sterility in the CR16‐knockout mice. The sperms of the CR16‐knockout mice had abnormal head morphology, and greatly diminished fertilization ability in in vitro fertilization experiments. CR16 and N‐WASP were localized to the actin filaments at the Sertoli cell–spermatid junctions (SspJs). The level of N‐WASP but not the transcript was decreased in the testes and Sertoli cells of the CR16‐knockout mice. Therefore, CR16 and N‐WASP are suggested to play important roles in spermatogenesis.