Pleurotus citrinopileatus is an edible medicinal mushroom rich in biomolecules and thus has a high potential for use in formulating pharmaceutical and nutraceutical products. To test its effect on body weight and glucose control, we generated diet-induced obese (DIO) C57BL/6J male mice by feeding the mice a high-fat diet (60% fat) for 8 weeks and treating them with an ethanolic P. citrinopileatus extract (PCE) at either 200 or 500 mg/kg body weight for 12 additional weeks. The results showed that PCE significantly inhibited high-fat diet-induced weight gain, fat accumulation, and glucose intolerance in the DIO mice. Moreover, the PCE had a beneficial effect on liver and kidney function. On the basis of these results, we conclude that PCE is effective in the treatment of metabolic syndrome and thus could be a good candidate for use in future pharmaceutical or nutraceutical applications.