Objective: This study aims to establish an efficient and fast method for in vivo knockdown of endogenous genes in mouse testis. Methods: We designed and constructed the lentiviral vector (pSliencer:GFP:shSox30) along with the control vector (pSliencer:GFP:shScramble), and prepared high-titer lentiviral particles. These lentiviral particles were microinjected into the mouse testis at postnatal day 24. Immunofluorescence and histological analysis was performed to evaluate the functional consequences in mouse testis upon Sox30 knock down. Results: After the shSox30 lentiviral particles treatment, SOX30 protein was substantially decreased in mouse testis. Immunofluorescence of testis sections revealed that many spermatids were arrested at step 2-3. Histological examination of testis sections revealed that elongated spermatids were absent in seminiferous tubules at stage VII-VIII. Conclusion: shRNA-mediated lentivirus effectively decreases the expression level of target genes in mouse testis, providing a useful platform to knockdown endogenous genes in vivo.