ScopeThe aim of this study is to investigate the signaling pathways by which allyl isothiocyanate (AITC) reduces adipocyte differentiation and the efficacy of AITC in suppressing galectin‐12 levels as a therapeutic for high fat diet (HFD)‐induced obesity.Methods and resultsAITC presents anti‐adipogenic effects on 3T3‐L1 cells by decreasing lipid droplet accumulation in a dose‐dependent manner. AITC suppresses 3T3‐L1 differentiation into adipocytes by decreasing galectin‐12 expression and by downregulating key adipogenic transcription factors. AITC influences the expression of 3T3‐L1 pre‐adipocytes by modulating adipokine expression (leptin and resistin) and by regulating the protein kinase B (PKB/Akt)/cAMP response element‐binding protein (CREB) pathway. In HFD‐fed mice, oral administration of AITC reduces the body weight, accumulation of lipid droplets in the liver, and white adipocyte size.ConclusionIn summary, the results indicate that AITC inhibits adipocyte differentiation by suppressing galectin‐12 levels in 3T3L1 cells and has antiobesity effects in HFD‐fed mice.