Prostate cancer (PCa) is an endocrine tumor that presents distinct metabolic features associated with neoplastic development, namely in the transition from the androgen-dependent to the androgen-independent phenotype that characterizes advanced stages of prostate cancer. Recently, we have found that LNCaP (androgen-responsive) and PC3 (androgen-nonresponsive) PCa cells present distinct glycolytic metabolism profiles in consequence of altered gene expression and/or activity of glycolytic enzymes and transporters. Therefore, this study was designed aiming to examine the effect of androgens on gene expression of glucose transporters and glycolytic enzymes in androgen-responsive PCa cells. LNCaP cells were treated with 10 nM of 5α-dihydrotestosterone (DHT) for 12, 24, and 48 h. Glucose consumption and lactate production were determined spectrophotometrically using commercial kits. Expression of glucose transporters (GLUT1 and GLUT3), phosphofructokinase 1 (PFK1), lactate dehydrogenase (LDH) and monocarboxylate transporter (MCT4) mRNA and protein was analyzed by real-time PCR and Western Blot, respectively. LDH enzymatic activity also was determined. The obtained results demonstrated that androgens stimulation diminished the expression of both GLUT1 and GLUT3, and increased PFK levels. Also, the expression of LDH and MCT4 was diminished in LNCaP cells in the presence of DHT, which was concomitant with decreased enzymatic activity of LDH. In addition, we verified that androgenic regulation of genes associated with glycolytic metabolism underlies altered glucose consumption and lactate production in LNCaP cells. These findings demonstrated that androgens are modulators of glycolytic metabolism in PCa cells, which may represent a relevant aspect driven prostate tumor development.