The exact function of the doublesex and mab-3 related transcription factor-like family a2 gene (dmrta2) has remained largely unknown possibly because of its functional redundancy with dmrta1 in most vertebrates. In this study, dmrta1 was demonstrated to likely be absent in the zebrafish genome, which facilitated our functional analysis of dmrta2 in this model organism. To analyze its gene function in embryos and adults, we generated a mutant form of Dmrta2 (R106Q, Dmrta2^RQ) with its in vitro DNA-binding capacity abolished and a transgenic line for the inducible expression of this mutant Dmrta2^RQ upon doxycycline (Dox) treatment. Preferential dmrta2 expression was detected in the developing brain during embryogenesis and in the adult testis. During embryogenesis, Dmrta2^RQ expression caused severe embryonic development defects and dramatic expression changes of two telencephalic marker genes, fibroblast growth factor 8a (fgf8a), and empty spiracles homolog 1 (emx1). In adults, the inducible Dmrta2^RQ expression occurred specifically in the adult testis and recapitulated the endogenous dmrta2 expression in this organ. Intriguingly, adult males expressing dmrta2^RQ showed normal spermatogenesis and were fertile, but the expression of cyclin-dependent kinase inhibitor 2C (cdkn2c), which is evolutionarily clustered with dmrta2, was significantly suppressed during spermatogenesis. Further protein-binding and promoter mutation analysis indicated that a putative Dmrta2-binding site on the cdkn2c promoter was required for sustaining the normal expression of cdkn2c during zebrafish spermatogenesis, suggesting that Dmrta2 might regulate the expression of cdkn2c.