Sertoli cells (SCs) glucose metabolism is crucial for spermatogenesis since developing germ cells consume lactate produced by SCs as their main energy source. Recently, androgens and estrogens have been implicated in SCs energy metabolism modulation, although the molecular mechanisms remained undisclosed. Here, we report the effect of sex steroid hormones on key points of cultured rat SCs glycolytic pathway. We used primary cultures of immature rat SCs treated with 17β-estradiol (E2) or 5α-dihydrotestosterone (DHT). The transcript levels of glucose transporters (GLUTs), phosphofructokinase 1 (PFK-1) and lactate dehydrogenase C (LDH C) were analyzed after 25 and 50 h of culture by qPCR. Protein levels of GLUTs, PFK-1, LDH and monocarboxylate transporter 4 (MCT4) after 25 and 50 h were determined by western blot and LDH activity was also assessed. Our results show that both E2 and DHT downregulated the transcript levels of PFK-1, GLUT1 and GLUT3 after 50 h. However, only DHT-treated cells presented a downregulation of LDH C transcript levels. Interestingly, the protein levels of these enzymes and transporters remained unaltered except in DHT-treated cells that presented a significant decrease on GLUT1 protein levels evidencing a possible site for the regulation of SCs glucose metabolism by androgens. Taken together, our results provide evidence that sex steroid hormones action in SCs energy metabolism is mediated through modulation in glycolysis-related transporters and enzymes, particularly at the transcriptional level. DHT decreased GLUT1 protein levels and increased LDH activity after 25 h, evidencing key points for this hormone action in the regulation of SCs metabolism.