ABSTRACT:It has been reported the presence of components from the coagulation thrombin‐generating pathway and prostaglandins (PGs) in human ovarian follicular fluid (FF) but with unclear functions. Moreover, thrombin can induce COX‐2 expression linking to PG synthesis in several cell types. Therefore, this study sought to explore the thromboxane (TX) generation in FF and the correlation between FF TX levels and oocyte maturation. The FF TXB2 (a stable metabolite of TXA2) levels exhibited a negative correlation between large preovulatory leading and small mid‐antral follicles from in vitro fertilization (IVF) patients, indicating a requirement of TX for a small follicle/oocyte to grow/mature. Further receiver operating characteristic curve analysis identified that intrafollicular TXB2 level could predict oocyte maturity. Thrombin was found expressed in a similar pattern/trend to TXB2 in intrafolliclar FF, where it could induce TXB2 production/secretion in human ovarian follicular GCs via proteinase‐activated receptor‐2 (PAR‐2) and PAR‐3 cooperation/transactivation and Erk/p38 MAPK/JNK signaling to cause COX‐2/TXB2 induction. Accordingly, PAR‐2 and ‐3 were present in human ovarian follicular GCs and thrombin, PAR‐2 agonist, and TXA2 analog caused a substantial enhancement in follicle development, which could be blocked by the PAR‐2, COX‐2, and TXA2 prostanoid (TP) receptor inhibitors in an ex vivo cultured murine ovary model. Collectively, we first demonstrated that FF thrombin regulates PAR‐2/‐3 cooperation and MAPK signaling pathways to induce COX‐2 expression and TX production in follicular GCs, possibly supporting intrafollicular TX levels and triggering TP‐related pathways to enhance follicle development. The FF TX levels also can be a predictor for oocyte maturation during IVF.