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The micro-environment of spermatogenesis is important for the improvement of in vitro fertilization (IVF). Therefore, developing a co-culture system may be valuable to improve the rate of IVF. In this study, we aimed to investigate the secretions of testicular sertoli cells (SCs) to find whether it can improve the micro-environment of IVF, by which promote the efficiency of fertilization in mice. The results showed that the motility of sperms in CCSCF group (sperms co-culture with SCs) was significantly promoted and the rate of fertilization were significantly increased compared with the CTR group (control group: sperms not co-culture with SCs). Moreover, we found that the estrogen concentrations, the expression of estrogen receptor (ER) and the phosphorylation of AMPK in sperms were higher in the CCSCF group than in CTR group. In all, our results indicated that SCs co-cultured with sperms can improve the motility of sperms, E2 secreted by SCs can increase Ca2+ level in the intracellular and the level of phosphorylation of AMPK through Ca-MKKβ in sperms. • The SCs co-culture with sperms to improve the micro-environment of IVF, by which promote the efficiency of fertilization. • SCs co-cultured with sperms may promote the motility of sperms. • E2 secreted by SCs increase the level of phosphorylation of AMPK through Ca-MKKβ. [ABSTRACT FROM AUTHOR]