The LH surge promotes terminal differentiation of follicular cells to become luteal cells. RUNX2 has been shown to play an important role in cell differentiation, but the regulation of Runx2 expression and its function in the ovary remain to be determined. The present study examined 1) the expression profile of Runx2 and its partner CBFbeta during the periovulatory period, 2) regulatory mechanisms of Runx2 expression, and 3) its potential function in the ovary. Runx2 expression was induced in periovulatory granulosa cells of human and rodent ovaries. RUNX2 and core binding factor-beta (CBFbeta) proteins in nuclear extracts and RUNX2 binding to a consensus binding sequence increased after human chorionic gonadotropin (hCG) administration. This in vivo up-regulation of Runx2 expression was recapitulated in vitro in preovulatory granulosa cells by stimulation with hCG. The hCG-induced Runx2 expression was reduced by antiprogestin (RU486) and EGF-receptor tyrosine kinase inhibitor (AG1478), indicating the involvement of EGF-signaling and progesterone-mediated pathways. We also found that in the C/EBPbeta knockout mouse ovary, Runx2 expression was reduced, indicating C/EBPbeta-mediated expression. Next, the function of RUNX2 was investigated by suppressing Runx2 expression by small interfering RNA in vitro. Runx2 knockdown resulted in reduced levels of mRNA for Rgc32, Ptgds, Fabp6, Mmp13, and Abcb1a genes. Chromatin immunoprecipitation analysis demonstrated the binding of RUNX2 in the promoter region of these genes, suggesting that these genes are direct downstream targets of RUNX2. Collectively, the present data indicate that the LH surge-induced RUNX2 is involved in various aspects of luteal function by directly regulating the expression of diverse luteal genes.