LM23 is a gene with testis-specific expression in Rattus norvegicus. To reveal the function of LM23 in the testis, we used lentivirus-mediated RNA interference (RNAi) to knock down LM23 expression in a tissue-specific manner in vivo. A lentiviral vector expressing a short hairpin RNA (shRNA) targeting LM23 was microinjected into the efferent ducts of R. norvegicus testes. The expression of LM23 in the treated testes was significantly knocked down compared with controls. These LM23-shRNA testes contained germ cells arrested at the spermatocyte stage, and showed increased apoptosis and disregulation of some meiotic genes. The results demonstrate the validity of the RNAi approach for targeting LM23 and reveal that LM23 expression in the testis is crucial for meiosis during spermatogenesis in R. norvegicus.