The ovulatory LH surge induces rapid up-regulation of Cyp11a1 in granulosa cells (GCs) undergoing luteinization during ovulation. This study investigated in vivo whether epigenetic controls including histone modifications and DNA methylation in the promoter region are associated with the rapid increase of Cyp11a1 gene expression after LH surge. GCs were obtained from rats treated with equine chorionic gonadotropin (CG) before (0 h) and 4 h and 12 h after human (h)CG injection. Cyp11a1 mRNA levels rapidly increased after hCG injection, reached a peak at 4 hours, and then remained elevated until 12 hours. DNA methylation status in the Cyp11a1 proximal promoter region was hypomethylated and did not change at any of the observed times after hCG injection. Chromatin immunoprecipitation assays revealed that the levels of trimethylation of lysine 4 on histone H3 (H3K4me3), an active mark for transcription, increased, whereas the levels of H3K9me3 and H3K27me3, which are marks associated with repression of transcription, decreased in the Cyp11a1 proximal promoter after hCG injection. Chromatin condensation, which was analyzed using deoxyribonuclease I, decreased in the Cyp11a1 proximal promoter after hCG injection. Chromatin immunoprecipitation assays also showed that the binding activity of CAATT/enhancer-binding protein-β to the Cyp11a1 proximal promoter increased after hCG injection. Luciferase assays revealed that the CAATT/enhancer-binding protein-β-binding site had transcriptional activity and contributed to basal and cAMP-induced Cyp11a1 expression. These results suggest that changes in histone modification and chromatin structure in the Cyp11a1 proximal promoter are involved in the rapid increase of Cyp11a1 gene expression in GCs undergoing luteinization during ovulation.