Sertoli cells actively metabolize glucose that is converted into lactate, which is used by developing germ cells for their energy metabolism. Androgens and oestrogens have general metabolic roles that reach far beyond reproductive processes. Hence, the main purpose of this study was to examine the effect of sex hormones on metabolite secretion/consumption in primary cultures of rat Sertoli cells. Sertoli cell-enriched cultures were maintained in a defined medium for 50 h. Glucose and pyruvate consumption, and lactate and alanine secretion were determined, by 1H-NMR (proton NMR) spectra analysis, in the presence or absence of 100 nM E2 (17β-oestradiol) or 100 nM 5α-DHT (dihydrotestosterone). Cells cultured in the absence (control) or presence of E2 consumed the same amount of glucose (29±2 pmol/cell) at similar rates during the 50 h. After 25 h of treatment with DHT, glucose consumption and glucose consumption rate significantly increased. Control and E2-treated cells secreted similar amounts of lactate during the 50 h, while the amount of lactate secreted by DHT-treated cells was significantly lower. Such a decrease was concomitant with a significant decrease in LDH A [LDH (lactate dehydrogenase) chain A] and MCT4 [MCT (monocarboxylate transporter) isoform 4] mRNA levels after 50 h treatment in hormonally treated groups, being more pronounced in DHT-treated groups. Finally, alanine production was significantly increased in E2-treated cells after 25 h treatment, which indicated a lower redox/higher oxidative state for the cells in those conditions. Together, these results support the existence of a relation between sex hormones action and energy metabolism, providing an important assessment of androgens and oestrogens as metabolic modulators in rat Sertoli cells.