Testis gene Znf230 may play a role in mammalian spermatogenesis according to previous reports. Deleting 5' important exons to block the formation of protein was a routine way in gene-knockout experiments. To investigate the physiological function of Znf230 gene, the mutant mice with disrupted exon 2 of Znf230 were generated in this study. Results showed that, mutant Znf230 mice were fertile and showed normal body, genitourinary organs, testes weights, and spermatid number but the litter size of the offspring reduced with unclear reasons. Hematoxylin and eosin staining showed that the testicular tissue of mutant mice was intact. Reverse transcriptase polymerase chain reaction analysis showed that two novel mutant transcripts appeared in the mutant mice: the short one including exon-1 and exon-3 to -6, the long one unexpectedly containing a partial sequence from the pPNT vector acting as a new exon 2. Bioinformatic analysis of the long transcript revealed that it might code a 24-kDa N-terminal mutant protein with the same 182 amino acids as that of the wild-type Znf230 in the C-terminus, indicating that the potential functional region of C3HC4-type RING finger was intact in mutant protein. Western blot and immunohistochemistry analyses also implied that this N-terminal mutation of Znf230 might not disrupt the possible role that wild-type Znf230 played in spermatogenesis. In summary, a potential exon structure in the targeting vector sequence involved in the expression of targeting Znf230 gene and disturbed the strategy of this gene-targeting experiment.