This study was undertaken to assess the effects of dehydroepiandrosterone (DHEA) administration and exercise training on muscular DHEA and 5α-dihydrotestosterone (DHT) levels and hyperglycemia in diet-induced obese and hyperglycemic rats. After 14 wk of a high-sucrose diet, obese male Wistar rats were assigned randomly to one of three 6-wk regimens: control, DHEA treatment, or exercise training (running at 25 m/min for 1 h, 5 days/wk; n = 10 each group). Results indicate that either 6 wk of DHEA treatment or exercise training significantly attenuated serum insulin and fasting glucose levels compared with the control group. Plasma and muscle concentrations of DHEA and DHT and expression levels of 5α-reductase were significantly higher in the DHEA-treated and exercise-training groups. Moreover, both DHEA administration and exercise training upregulated GLUT4 translocation with concomitant increases in protein kinase B and protein kinase Cζ/λ phosphorylation. Muscle DHEA and DHT concentrations closely correlated with blood glucose levels (DHEA treatment: r = -0.68, P < 0.001; exercise training: r = -0.65, P < 0.001), serum insulin levels, and activation of the GLUT4-regulated signaling pathway. Thus, increased levels of muscle sex steroids may contribute to improved fasting glucose levels via upregulation of GLUT4-regulated signaling in diet-induced obesity and hyperglycemia.