This study was conducted to evaluate the impact of deoxynivalenol (DON) and zearalenone (ZEA) metabolite, α-zearalenol (α-Zol), on cell proliferation and steroidogenesis of bovine large (LG) follicle granulosa cells (GC). LGGC were obtained from bovine ovarian follicles (8–22mm) and were cultured for 2 days in medium containing 10% fetal bovine serum followed by 1 or 2 days in serum-free medium without (control) or with treatments. Three different experiments were performed using different dosages of DON and α-Zol and in different combinations and a fourth experiment evaluated estradiol effects on granulosa cell proliferation. DON inhibited progesterone (P4) and estradiol (E2) production at high dose. α-Zol alone and in combination with DON increased cell growth. Estradiol inhibited cell growth indicating α-Zol is not acting as an estrogen agonist. This study demonstrates that α-Zol and DON can impact in vitro GC function, however further studies will be required to better understand the mechanism of action and reproductive effects of Fusarium mycotoxins.