Previous studies have suggested that vinegar intake can help to reduce body fat and hyperglycemia. Therefore, this study aimed to evaluate the anti-obesity efficacy of vinegar fermented using Cudrania tricuspidata fruits (CTFV) and its main phenolic constituents and to analyze its molecular mechanism and changes in obesity-related metabolizing enzymatic activities. We found that HFD significantly caused hepatic steatosis; increases in body fats, feed efficiency, liver mass, lipids, insulin, oxidative parameters, cardiovascular-associated risk indices, lipase and α-amylase activities, whereas CTFV efficaciously attenuated HFD-induced oxidant stress, fat accumulation, obesity-related enzymatic activity, and the activation or reduction of obesity-related molecular reactions via improving metabolic parameters including phosphorylated insulin receptor substrate 1, protein tyrosine phosphatase 1B, phosphorylated phosphoinositide 3-kinase/protein kinase B, phosphorylated mitogen-activated protein kinases, sterol regulatory element-binding protein 1c, CCAAT/enhancer-binding protein, and fatty acid synthase; and decreases in adiponectin receptor 1, leptin receptor, adenosine monophosphate-activated protein kinase, acetyl-CoA carboxylase, and peroxisome proliferator-activated receptor, subsequently ameliorating HFD-induced obesity. Therefore, CTFV might provide a functional food resource or nutraceutical product for reducing body fat accumulation.