Ovarian folliculogenesis involves a dynamic interplay of growth factors, hormones, follicle metabolism, and the physical environment of the ovary. Oxygen tension is critical for follicle growth and metabolism, especially for early-stage follicles where vascularity is limited. Understanding oxygen needs and metabolism in a multi-cellular organ such as the follicle, in which somatic cell numbers expand at a rapid rate, will make it possible to improve in vitro ovarian follicle growth and maturation. In this study, early secondary (100 μm) murine follicles were isolated and encapsulated in an alginate hydrogel to replicate the in vivo environment of the growing/maturing follicle. Encapsulated follicles were cultured for 8 days at either 2.5% O₂ or 20% O₂. Survival (2.6-fold) and growth (1.2-fold) were significantly higher for follicles cultured at 2.5% O₂ compared to 20% O₂. Using a mouse hypoxia signaling pathway qRT-PCR array and GeneGo Metacore analysis, we found that direct target genes of hypoxia-activated HIF1 complex were significantly up-regulated in follicles cultured for 8 days at 2.5% O₂ comparedwith follicles cultured at 20% O₂, including the carbohydrate transport and metabolism genes Slc2a3, Vegfa, Slc2a1, Edn1, Pgk1, Ldha, and Hmox1. Other up-regulated genes included carbohydrate transporters (Slc2a1, Slc2a3 and Slc16a3) and enzymes essential for glycolysis (Pgk1, Hmox1, Hk2, Gpi1, Pfkl, Pfkp, Aldoa, Gapdh, Pgam1, Eno1, Pkm2 and Ldha). For follicles cultured at 2.5% O₂, a 7.2-fold up-regulation of Vegfa correlated to an 18-fold increase in VEGFA levels in the culture media and a 3.2-fold up-regulation of Ldha correlated to a 4.8-fold increase in lactate levels in the culture media. Both VEGFA and lactate levels were significantly up-regulated in follicles cultured at 2.5% O₂ compared to 20% O₂. Together, these data suggest that enhanced hypoxia-mediated glycolysis increases growth and survival of early follicles, and provides key insights into early follicle metabolism in the ovary.