Female animals living in the Qinghai-Tibet Plateau have lower ovulation rates because of the hypoxic environment, however, the mechanism of hypoxia on animal follicles is unclear. In this study, the effects of hypoxia on bovine follicles were investigated using an in vitro follicular culture system. The results show that there was a significant decrease in follicular diameter from day 3 to day 6 in both hypoxia and hypoxia with estrogen (E) and fulvestrant (ICI 182780) (hypoxia + E + ICI) groups, when compared with a normoxia group ( < 0.05). We also observed significant downregulation of ERα and SHR while upregulation of LHCGR expression in the hypoxia group and hypoxia + E + ICI groups compared to the normoxia group ( < 0.05). The expression of IGF1 gene was significantly downregulated in hypoxia + E + ICI group when compared to the hypoxia + E group ( < 0.05). The expression of HIF1A, ADAMTS1, VEGFA, and EDN2 were upregulated in both hypoxia and hypoxia + E + ICI groups in comparison to normoxia group ( < 0.05). Under hypoxic conditions, the addition of E resulted in a decrease of HIF1A protein but an increase of ERα protein in cultured bovine follicles ( < 0.05). In summary, hypoxia limits the growth of bovine follicle cultured in vitro through inhibition of ERα.