Obesity is a major health concern in modern societies as it is linked to diverse chronic diseases, such as diabetes, cancer, stroke, and skeletomuscular disorders. This study aimed to investigate the lipolytic potency of the metabolic suppressor 3-iodothyronamine (T1AM) and its molecular mechanism in differentiated 3T3-L1 adipocytes. Cells stained with Oil Red O showed a remarkable accumulation of lipid droplets by 20-d post-differentiation and a plateau at 26 - 30 day. Treatment with 100 μM T1AM for 6 h increased the liberation of free fatty acids (FFAs) and glycerol (P < 0.05) detected in the culture media. However, this stimulatory effect was significantly suppressed by ATGListatin, an inhibitor of adipose triglyceride lipase (ATGL), suggesting that ATGL plays a rate-limiting role in triglyceride (TG) turnover. To understand the lipolytic mechanism, immunoblotting and confocal image analyses of the T1AM-treated and control groups were conducted. The elevated lipolysis was accompanied by increases in the phosphorylation of adenosine monophosphate-activated protein kinase (p-AMPK), nuclear localization of forkhead box O1 (FoxO1), and expression of monoacylglycerol lipase (MGL) protein (P < 0.05). Finally, the treated cells exhibited downregulated expression of acetyl-CoA carboxylase (ACC) relative to p-ACC and increased protein expression of carnitine palmitoyltransferase 1 (CPT1) (P < 0.05). Taken together, T1AM showed lipolytic potency via activation of the AMPK/FoxO1/ATGL/MGL axis for decomposing TGs to FFAs and glycerol and of the AMPK/ACC/CPT1 pathway in facilitating the mobilization of FFAs into the mitochondria, highlighting its therapeutic potential for the treatment of obesity.