Activating transcription factor 6 (ATF6) is an important factor in the endoplasmic reticulum (ER) stress signaling pathway. The aim of this study was to assess the role of ATF6 in mouse granulosa cells with respect to apoptosis, the cell cycle, and steroid hormone production, as well as several key genes related to follicular development, via RNA interference, immunohistochemical staining, real-time quantitative polymerase chain reaction, western blotting, flow cytometry, TUNEL assay, and ELISA. Immunohistochemical staining revealed that ATF6 was extensively distributed in the granulosa cells of various ovarian follicles and oocytes in adult female mice. FSH or LH treatment significantly increased ATF6 protein levels .Flow cytometry and TUNEL assays analysis indicated that ATF6 depletion decreased apoptosis and arrested the S-phase of cell cycle. Consistent with these results, p53, Caspase-3, Bax, Chop, CyclinA1, CyclinB1, and CyclinD2 mRNA expression decreased, whereas Bcl-2 and Grp78 mRNA expression increased. Interestingly, ATF6 knock-down obviously increased progesterone and estradiol production. Cyp1b1 mRNA levels were down-regulated, whereas Cyp11a1, Star and Cyp19a1 mRNA levels were up-regulated, in keeping with the changes in steroid hormones. Furthermore, ATF6 disruption remarkably increased Igfbp4 expression and decreased Has2, Ptgs2 and Ptgfr expression. But after treating with tunicamycin (Tm), the levels of Has2, Ptgs2 and Ptgfr increased relatively whereas Igfbp4 expression decreased. Collectively, these results imply that ATF6 may regulate apoptosis, the cell cycle, steroid hormone synthesis, and other modulators related to folliculogenesis, which may indirectly involve in the development, ovulation, and atresia of ovarian follicles by affecting the physiological function of granulosa cells.