Lemon (Citrus limon) has antioxidant, immunoregulatory, and blood lipid-lowering properties. This study aimed to determine the effect of the lemon fermented product (LFP) which is lemon fermented with Lactobacillus OPC1 to prevent obesity. The inhibition of lipid accumulation in 3T3-L1 adipocytes is examined using a Wistar rat model fed a high-fat diet to verify the anti-obesity efficacy and mechanism of LFP. Here, it was observed that LFP reduced cell proliferation and inhibited the lipid accumulation (8.3%) of 3T3-L1 adipocytes. Additionally, LFP reduced body weight (9.7%) and fat tissue weight (25.7%) of rats; reduced serum TG (17.0%), FFA (17.9%), glucose (29.3%) and ketone body (6.8%); and increased serum HDL-C (17.6%) and lipase activity (17.8%). LFP regulated the mRNA expression of genes related to lipid metabolism (PPARγ, C/EBPα, SREBP-1c, HSL, ATGL, FAS, and AMPK). Therefore, LFP reduces body weight and lipid accumulation by regulating the mRNA expression of genes related to lipid metabolism. Overall, our results implicate LFP as a potential dietary supplement for the prevention of obesity.