The present study investigated the anti-obesity effects of Mycoleptodonoides aitchisonii (MA) in mice fed a high-fat (HF) diet. Two groups were fed either a normal control diet or an HF (45% kcal fat) diet for 12 weeks and three groups were fed an HF diet supplemented with powdered MA (MAP, 1%, 3%, and 5%) for 12 weeks. The anti-obesity effects of MAP supplementation on body weight, fat mass development, and lipid-related markers were assessed. Consumption of an HF diet resulted in increased body weight, serum lipids, relative adipose tissues weight, and liver fat accumulation. However, administration of MAP significantly decreased body weight gain, food intake, food efficiency ratio, hepatic cholesterol level, and adipose tissue weight in a dose-dependent manner. In addition, treatment with MAP significantly reduced the occurrence of fatty liver deposits and steatosis, and inhibited an HF diet-induced increase in adipocyte size. These results suggest that dietary supplementation with MAP exerts anti-obesity effects and indicate that MAP could be used as a functional food to control obesity.