AbstractInduction of the brown adipocyte‐like phenotype in white adipocytes (browning) is considered as a novel strategy to fight obesity due to the ability of brown adipocytes to increase energy expenditure. Here, we report that L‐rhamnose induced browning by elevating expression levels of beige‐specific marker genes, including Cd137, Cited1, Tbx1, Prdm16, Tmem26, and Ucp1, in 3T3‐L1 adipocytes. Moreover, L‐rhamnose markedly elevated expression levels of proteins involved in thermogenesis both in 3T3‐L1 white and HIB1B brown adipocytes. L‐rhamnose treatment in 3T3‐L1 adipocytes also significantly elevated protein levels of p‐HSL, p‐AMPK, ACOX, and CPT1 as well as reduced levels of ACC, FAS, C/EBPα, and PPARγ, suggesting its possible role in enhancement of lipolysis and lipid catabolism as well as reduced adipogenesis and lipogenesis, respectively. The quick technique of efficient molecular docking provided insight into the strong binding of L‐rhamnose to the fat‐digesting glycine residue of β3‐adrenergic receptor (AR), indicating strong involvement of L‐rhamnose in fat metabolism. Further examination of the molecular mechanism of L‐rhamnose revealed that it induced browning of 3T3‐L1 adipocytes via coordination of multiple signaling pathways through β3‐AR, SIRT1, PKA, and p‐38. To the best of our knowledge, this is the first study to demonstrate that L‐rhamnose plays multiple modulatory roles in the induction of white fat browning, activation of brown adipocytes, as well as promotion of lipid metabolism, thereby demonstrating its therapeutic potential for treatment of obesity. © 2018 IUBMB Life, 70(6):563–573, 2018