The testis specific serine/threonine protein kinase family (Tssk) members play important roles in spermatogenesis and/or spermiogenesis. Similar to other Tssk family members, Tssk4 protein shows exclusive expression in testis, but its biochemical and biological functions are still largely unknown. In present work, we generate a polyclonal antibody which specifically recognizes Tssk4 but not the other three Tssk family members (Tssk1, Tssk2 and Tssk3). By using the qualified antibody, we show that Tssk4 protein is constantly expressed in testis from haploid round spermatids to morphological mature spermatozoa. Further experiments reveal that Tssk4 has autophosphorylation activity and self-association character in vitro. Importantly, we find that autophosphorylation of Tssk4 at Thr-197 in the T-loop region is essential to its kinase activity. Taken together, these findings suggest that autophosphorylation at Thr-197 plays a critical role in maintaining Tssk4 kinase activity, which might be involved in spermiogenesis.