Spermatogonial Stem Cells (SSCs) are the basis of spermatogenesis and male fertility. SSCs hold a tremendous potential for the treatment of infertility, through the biobanking of testicular tissue and subsequent isolation, expansion in culture, and transplantation of SSCs to replenish spermatogenesis; laboratory techniques that require a profound knowledge of SSC metabolic requirements. While mouse SSC precursors, or primordial germ cells (PGCs), rely on oxidative phosphorylation (OXPHOS), adult human and mouse SSCs mainly use glycolysis to maintain SSC-specific circuits. The metabolic phenotype of prepubertal SSCs and the timeline of metabolic transitions during juvenile development have not been described in any species. Using the pig model, we show that early prepubertal spermatogonia rely on OXPHOS and transition to an adult-like SSC metabolism with 2 months of age, associated with an upregulation of SSC-specific signaling pathways. We delineated a timeline of human SSC metabolic development, from birth to puberty, using single-cell RNA sequencing and deciphered that prepubertal SSCs show a high expression of OXPHOS-related genes until 11 years of age. We further revealed that niche polarization events, visible as tight junction formation between adjacent Sertoli cells, coincide with the maturation of SSC morphology at 11 years that preceded their metabolic transition, and are associated with an accumulation of lipid droplets in Sertoli cells. The accumulation of lipid droplets was conserved between the species. Lipidomic and quantitative (phospho-) proteomic analysis of porcine Sertoli cells revealed a drastic increase of triglycerides (TGs) and decrease of sphingolipids and discovered altered proteins and phosphorylation-sites associated with androgen receptor signaling, cytoskeletal remodeling, and lipid metabolism with maturation, respectively. Together, this work shows that the prepubertal development of SSCs is characterized by distinct metabolic transitions, which require state-specific culture conditions. An accumulation of lipid droplets could indicate Sertoli cell maturational events as critical point in the formation of the SSC niche microenvironment and therefore serve as an indirect tissue-based biomarker for SSC metabolic maturation. This knowledge provides a pivotal piece in understanding the prepubertal development of SSCs and associated niche maturation events that will help to ultimately facilitate their clinical use.