Spermatogonial stem cells (SSCs) are a pivotal cell population responsible for maintaining lifelong spermatogenesis, however, gaps in knowledge exist in our understanding of the metabolic requirements for SSC maintenance. Recently, our research group identified that SSCs reside in hypoxic microenvironments in the testis (Bernstein et al., 2023; under review). We hypothesised that this low oxygen availability would drive glycolytic metabolism and be intertwined with SSC maintenance, as is the case with other adult stem cell types. A first-pass single-cell RNA sequencing analysis of mouse and human spermatogonia confirmed that SSCs possess a transcriptomic signature reminiscent of a cell residing in hypoxia and utilising glycolytic metabolism (e.g., Aldoa, Pdk2, Gapdh, Myc), while that of progenitor and differentiating spermatogonia reflected mitochondrial biogenesis and oxidative phosphorylation (e.g., Ndufb7, Polg, Opa1, Cox11). Follow-up analyses assessing mitochondrial copy number in SSCs and progenitors (isolated from neonatal ID4-EGFP transgenic mice) corroborated this, identifying an approaching-significant increase in copy number (p=0.0639) upon the SSC-to-progenitor transition. To explore any interconnection between these metabolic preferences and a hypoxic state, we employed pharmacological stabilisation of hypoxia-inducible factors using a prolyl hydroxylase inhibitor, Daprodustat. Daprodustat treatment of undifferentiated spermatogonia in culture decreased oxygen consumption and increased extracellular acidification rates indicative of a shift towards glycolytic metabolism. Importantly, Daprodustat treatment significantly improved the maintenance of SSCs in vitro (p<0.05), as demonstrated by a spermatogonial transplantation assay. These findings highlight a pivotal role for hypoxia in mediating metabolic adaptation and self-renewal of SSCs and suggest that mitochondrial biogenesis and a shift to aerobic metabolism are important components of spermatogonial differentiation. Additionally, this research provides compelling evidence for the therapeutic potential of manipulating hypoxic pathways to maintain and manipulate SSCs in vitro. This could have direct implications for the development of stem cell therapies aimed at reversing male infertility.