Obesity has brought great challenges to global human health, and how to effectively prevent and control the occurrence and development of obesity has become an urgent problem. The role and mechanism of 4-[(α-Lrhamnosyloxy) benzyl] isothiocyanate (MITC), an active ingredient of Moringa oleifera Lam., in the regulation of lipid metabolism have not been comprehensively investigated. In the present study, we investigated the mechanism of MITC in inhibiting lipid accumulation in mice fed with a high-fat diet (HFD) in terms of both lipolysis and central appetite regulation mediated by the gut microbe-gut-brain axis. MITC enhanced the characteristic indices associated with HFD mice and also promoted adipocytolysis and brown fat thermogenesis. Moreover, MITC was observed to improve leptin resistance, modulate the composition of gut microbiota such as Ruminococcaceae, Parasutterella, and Acetatifactor, promote 5-hydroxytryptamine secretion, further enhance the secretion of glucagon-like peptide-1 (GLP-1) and peptide tyrosine-tyrosine (PYY) to activate peroxisome proliferator-activated receptor (PPAR) signaling in the hypothalamus, and modulate feeding behavior to inhibit lipid accumulation in HFD mice. These data suggest that MITC supplementation can help to alleviate obesity or obesity-related diseases.