Phosphorylation on serine/threonine or tyrosine residues of target proteins is an essential and significant regulatory mechanism in signal transduction during many cellular and life processes, including spermatogenesis, oogenesis and fertilization. In the present work, we reported the isolation and characterization of mouse testis-specific serine/threonine kinase 5 (Tssk5), which contains four alternatively spliced variants including, Tssk5alpha, Tssk5beta, Tssk5gamma and Tssk5delta. Moreover, the locus of Tssk5 is on chromosome 14qC3 and the four variants had a similar high expression in the testis and the heart; however, had a low expression in other tissues, except for Tssk5alpha which also had comparably high expression in the spleen. Each variant of Tssk5 expression began in the testis 16 days after birth. Aside from TSSK5alpha, the other isoforms have an insertion of ten amino acid residues (RLTPSLSAAG) in region VIb (HRD domain) (His-Arg-Asp). Moreover, only TSSK5alpha exhibited kinase activity and consistently, a further Luciferase Reporter Assay demonstrated that TSSK5beta, TSSK5gamma and TSSK5delta cannot be stimulated at the CREB/CRE responsive pathway in cmparison to TSSK5alpha. These findings suggest that TSSK5beta, TSSK5gamma, TSSK5delta may be pseudokinases due to the insertion, which may damage the structure responsible for active kinase activity. Pull-down assay experiments indicated that TSSK5beta, TSSK5gamma and TSSK5delta can directly interact with TSSK5alpha. In summary, these four isoforms with similar expression patterns may be involved in spermatogenesis through a coordinative way in testis.