Testosterone is a male sex hormone and low circulating testosterone levels are associated with various health disorders in men. Obesity results in reduced circulating testosterone levels in men. Previously, we demonstrated that lifestyle modifications (combination of aerobic exercise and dietary modification) increase circulating testosterone levels in overweight/obese men. However, the effect of regular aerobic exercise on serum testosterone levels remains unclear. The purpose of this study was to investigate the effect of a 12-week aerobic exercise intervention on circulating testosterone levels in normal-weight and overweight/obese men. Sixteen normal-weight men and twenty-eight overweight/obese men completed a 12-week aerobic exercise intervention. Before and after the intervention, we measured serum total testosterone, free testosterone, and bioavailable testosterone levels, and categorized the physical activity levels (light, moderate, or vigorous) in all participants. At baseline, serum total testosterone, free testosterone, and bioavailable testosterone levels were significantly lower in overweight/obese men than in normal-weight men (all p<0.01). After the 12-week aerobic exercise intervention, serum total testosterone, free testosterone, and bioavailable testosterone levels significantly increased in overweight/obese men (p<0.01). In addition, stepwise multivariable linear regression analysis revealed the increase in vigorous physical activity was independently associated with increased serum total testosterone levels (β=0.47, p=0.011). We demonstrated that a 12-week aerobic exercise intervention increased serum total testosterone, free testosterone, and bioavailable testosterone levels in overweight/obese men. We suggest that an increase in vigorous physical activity increased circulating testosterone levels in overweight/obese men.