Obesity is defined as excess adipose mass that causes serious health problems. Phenethyl isothiocyanate (PEITC) is a major and relatively nontoxic compound of the isothiocyanates. Although many studies have demonstrated that PEITC is a potent substance with physiological activities, such as anticancer activity, the precise mechanism for the effects of PEITC on inflammation and lipid metabolism in adipose tissue is not clear. Our study aimed to clarify the effects of PEITC supplements on the adipose tissue in obesity induced with a high-fat/cholesterol diet, and the underlying mechanisms. We induced obesity by feeding the mice with high fat with 1% cholesterol diet (HFCD) for 13 weeks. Mice were divided into five groups: normal diet (CON), HFCD, HFCD with 3 mg/(kg·d) gallic acid (HFCD+G), and HFCD with 30 and 75 mg/(kg·d) PEITC (HFCD+P30 and HFCD+P75, respectively). Using western blotting and quantitative polymerase chain reaction (qPCR) analysis of the adipose tissue, we determined the expression of lipid metabolism-related genes and inflammation-related genes. In the HFCD, the expression level of nuclear factor-κB (NF-κB), lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), and cyclooxygenase-2 (COX-2), was higher compared with that in the CON. Moreover, in the HFCD, the expression of p-mechanical targets of the rapamycin (mTOR) was increased, whereas that of p-AMP-activated protein kinase (AMPK) was decreased compared with that in the CON. Nevertheless, these decreased expression levels of p-AMPK and increased levels of LOX-1, p-mTOR, peroxisome proliferator-activated receptor gamma (PPARγ), NF-κB, and COX-2, were alleviated by PEITC supplementation. Therefore, we suggest that PEITC might be a potential preventive agent for ameliorating obesity-induced inflammation and adipogenesis by modulating the mTOR/AMPK/PPARγ pathway.