〔Abstract〕 To verify the association between the Ddit3-Trib3-Akt signaling pathway and rat spermatogenesis by constructing an in vitro co-culture system of testis. Methods Testicular tissue blocks from 20 ~ 25-day-old male rats were placed in an in vitro culture system, and the culture medium was replaced every 2 to 3 days. PCR was used to verify the expression of marker genes of various spermatogenic cells. RNA interference technology was employed to verify the correlation between the Ddit3-Trib3-Akt signaling pathway and rat spermatogenesis. Results The co-culture system could be continuously cultured for more than 2.5 months in vitro. RT-PCR showed that specific marker genes of spermatogonia, spermatocyte and spermoblast were expressed. The RNA and protein expression ofTrib3 and Akt changed after the knocking down of Ddit3 and Trib3, respectively. It demonstrated the existence of Ddit3-Trib3-Akt signaling pathway in rat spermatogenesis. Conclusion The culture time of more than 2.5 months indicates that the culture system can temporarily maintain the proliferation and differentiation of stem cells, and simultaneously maintain and stabilize spermatogenesis in a simple system. The successful validation of the Ddit3-Trib3-Akt signaling pathway also confirms that this culture system can be used to study possible molecular mechanisms of spermatogenesis in vitro.