Core binding factor β (CBFβ) is a non-DNA-binding partner of all RUNX proteins and critical for transcription activity of CBF transcription factors (RUNXs/CBFβ). In the ovary, the expression of Runx1 and Runx2 is highly induced by the luteinizing hormone (LH) surge in ovulatory follicles, whereas Cbfb is constitutively expressed. To investigate the physiological significance of CBFs in the ovary, the current study generated two different conditional mutant mouse models in which granulosa cell expression of Cbfb and Runx2 was reduced by Cre recombinase driven by an Esr2 promoter. Cbfbgc-/- and Cbfbgc-/- × Runx2gc+/- mice exhibited severe subfertility and infertility, respectively. In the ovaries of both mutant mice, follicles develop normally, but the majority of preovulatory follicles failed to ovulate either in response to human chorionic gonadotropin administration in pregnant mare serum gonadotropin-primed immature animals or after the LH surge at 5 months of age. Morphological and physiological changes in the corpus luteum of these mutant mice revealed the reduced size, progesterone production, and vascularization, as well as excessive lipid accumulation. In granulosa cells of periovulatory follicles and corpora lutea of these mice, the expression of Edn2, Ptgs1, Lhcgr, Sfrp4, Wnt4, Ccrl2, Lipg, Saa3, and Ptgfr was also drastically reduced. In conclusion, the current study provided in vivo evidence that CBFβ plays an essential role in female fertility by acting as a critical cofactor of CBF transcription factor complexes, which regulate the expression of specific key ovulatory and luteal genes, thus coordinating the ovulatory process and luteal development/function in mice.