Cyclic AMP response element-binding protein 1 (CREB1), a member of the CREB family, is known to be involved in follicular growth, ovulation, and ovarian disease. However, the physiological function of CREB1 in mouse granulosa cells (mGCs) remains lagerly unknown. The aim of this study was to determine the role of CREB1 in mGCs by knocking down CREB1 expression. CREB1 knock-down in mGCs at the mRNA and protein levels, was confirmed by quantitative real-time polymerase chain reaction and western blot. Results of enzyme linked immunosorbent assay revealed that CREB1 knockdown significantly decreased the concentrations of estradiol (E2) and progesterone (P4) in mGCs. Furthermore, the CREB1 knockdown in mGCs promoted cell proliferation and apoptosis, and arrested the cell cycle in S-phase. To elucidate the regulatory mechanism underlying the effects of CREB1 knockdown on steroid synthesis, cell cycle, and apoptosis, we measured the protein expression levels of several related genes in mGCs knocked down CREB1. When CREB1 was knocked down, the expression of Cyp1b1 and Cyp19a1, which encode steroidogenic enzymes, was down-regulated; the expression of the cell cycle factors CyclinA1, CyclinB1, and CyclinD2 were significantly decreased. Among apoptosis-related genes, Bcl-2 was down-regulated, whereas Bax and cleaved Caspase3 were upregulated. Moreover, CREB1 knockdown significantly decreased expression level of Has2, Ptgs2, and Igfbp4, which are essential genes for folliculogenesis in mGCs. Taken together, these findings suggested that CREB1 might be a key regulator of mGCs through regulating steroid synthesis, cell proliferation, cell cycle, apoptosis, and other regulators of folliculogenesis.