Colchicine has been used for therapeutic purposes and has attracted considerable attention because of its association with tubulin and the inhibition of small tubular polymerization. Although several studies have examined the possible preventive role of colchicine in metabolic diseases, its role in adipocytes is largely unknown. This study examined the novel functional role of colchicine in adipocytes demonstrating that colchicine stimulates browning in cultured white adipocytes. Colchicine stimulates browning by increasing the brown- and beige fat-specific markers in 3T3-L1 white adipocytes. Interestingly, colchicine decreased the expression of the main lipolytic proteins (ATGL, p-HSL) while it activated Ces3, suggesting a possibility for supplying essential fatty acids for inducing thermogenesis. Molecular docking analysis showed that colchicine has a strong affinity against GABA-BR and I33-AR, and its binding activity with GABA-BR (-26.52 kJ/mol) was stronger than I33-AR (-20.71 kJ/mol). Mechanistic studies were conducted by treating the cells separately with agonists and antagonists of GABA-BR and I33-AR to understand the molecular mechanism underlying the browning effect of colchicine. The results showed that colchicine stimulates browning via the antagonism of GABA-BR and the agonism of I33-AR in 3T3-L1 white adipocytes. The colchicine-mediated activation of I33-AR stimulated the PKA/ p38 MAPK signaling pathway, where consequently ATF2 acted as a positive regulator, but AFT4 was a negative regulator for the induction of browning.