Cytochrome P450 lanosterol 14-demethylase (CYP51) is a key enzyme in sterols and steroids biosynthesis that can induce meiotic resumption in mouse oocytes. The present study investigated the expression mechanism and function of CYP51 during FSH-induced mouse cumulus oocyte complexes (COCs) meiotic resumption. FSH increased cAMP-dependent protein kinase (PKA) RII level, induced cAMP response element binding protein (CREB) phosphorylation and CYP51 expression in cumulus cells prior to oocyte meiotic resumption. Moreover, CYP51 and epidermal growth factor (EGF)-like factor, amphiregulin (AR) expression were blocked by KG-501 (a drug interrupting the formation of CREB functional complex). KG-501 and RS21607 (a specific inhibitor of CYP51 activity) inhibited oocyte meiotic resumption, which can be partially rescued by progesterone. These two inhibitors also inhibited FSH-induced MAPK phosphorylation. EGF could rescue the suppression by KG-501 but not RS21607. Furthermore, type II PKA analog pairs, N⁶-cAMP plus 8-Br-cAMP, increased PKA RII level and mimiced the action of FSH, including CREB phosphorylation, AR and CYP51 expression, MAPK activation and oocyte maturation. All these data suggest that CYP51 plays a critical role in FSH-induced meiotic resumption of mouse oocytes. CYP51 and AR gene expression in cumulus cells are triggered by FSH via a type II PKA/CREB dependent signal pathway. Our study also implicates that CYP51 activity in cumulus cells participates in epidermal growth factor receptor (EGFR) signaling regulated oocyte meiotic resumption.