The PDZ-binding kinase (PBK) protein is localised exclusively in spermatogenic cells, such as spermatogonia, spermatocytes and round spermatids, of the adult testis. However, its role in male fertility remains unknown. Analysis of adult Pbk-knockout (KO) male mice showed no significant difference in the weight of the testes, epididymis and seminal vesicle compared with adult wild-type (WT) mice. There were no significant differences in testis morphology, tubule diameter and the number of offspring born to females mated with KO or WT male mice. Sperm number, motility and morphology did not differ significantly between KO and WT mice. The oocyte fertilisation rate and embryo development following IVF were comparable between groups fertilised using spermatozoa from KO versus WT mice (P>0.05). Further analysis revealed that the phosphorylation of the mitogen-activated protein kinases (MAPKs) p38 kinase, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinases was dysregulated in the testis of KO mice. In conclusion, Pbk-KO male mice are fertile and their spermatozoa and testis do not show any morphological and functional abnormalities despite the dysregulated phosphorylation of MAPKs. It is likely that functional redundancy of PBK and overlapping substrate specificities of the MAPK superfamily compensated for the loss of PBK from the testis.