Patients with obesity mostly have metabolic syndrome and this can lead to multiple health problems. In the present study, we evaluated the anti-obesity effect of water-soluble red pepper (Capsicum annuum L.) leaf extract (PLE) on 3T3-L1 adipocytes and high-fat diet (HFD)-fed mice. The adipocyte lipid content was determined using Oil Red O staining, which revealed that 100 μg mL-1 PLE markedly reduced fat accumulation without affecting the cell viability. PLE exhibited high prebiotic activity scores by modulating probiotic strains, contributing to host health improvement. In vivo investigation in HFD-fed mice revealed that PLE supplementation significantly decreased the HFD-induced increases in the body weight, amount of white adipose tissue, and serum triglyceride, total cholesterol, leptin, and insulin levels. Consistent with its effects on reduced lipid droplet formation in the liver, PLE supplementation suppressed the expression of lipid synthesis-related proteins including SREBP-1, FAS, and PPAR-γ in the liver and increased that of PGC-1α, CPT1, and adiponectin in epididymal WAT. PLE treatment improved intestinal barrier function and inflammation and reduced harmful intestinal enzyme activities in the feces. Collectively, these results indicate that PLE inhibits fat accumulation in HFD-fed mice via the suppression of adipogenesis and lipogenesis, suggesting its potential in preventing obesity.