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Obesity is associated with metabolic disorders. Thus, obesity prevention and treatment are essential for health. <i>Antrodia cinnamomea</i> (AC) is a multifunctional medicinal fungus used for the treatment of various diseases and for preventing diet-induced obesity. Leptin deficiency causes over-eating and spontaneous obesity. The concomitant metabolic symptoms are more severe than diet-induced obesity. Here, we used leptin-deficient (<i>ob/ob</i>) mice as an animal model for over-feeding to study the effect of AC on obesity. We fed C57BL/6 mice (WT, <i>ob+/+</i>) and <i>ob/ob</i> mice with AC for four weeks before performing qRT-PCR and immunoblot analysis to elaborate AC-modulated mechanisms. Further, we used Caco-2 cells as a human intestinal epithelial barrier model to examine the effect of AC on intestinal permeability. Our results suggested that AC reduces lipid deposits of the liver and epididymal white adipose tissue (EWAT) by promoting lipid metabolism and inhibiting lipogenesis-associated genes and proteins in <i>ob/ob</i> mice. Moreover, AC effectively repaired intestinal-barrier injury caused by leptin deficiency and enhanced intestinal barrier integrity in Caco-2 cells. Interestingly, AC significantly reduced body weight and EWAT with no compromise on food intake in <i>ob/ob</i> mice. Thus, AC effectively reduced obesity caused by leptin-deficiency and can potentially be used as a nutraceutical for treating obesity.