2’-Fucosyllactose (2’-FL), the functional oligosaccharide naturally present in milk, has been shown to exert health benefits. This study was aimed to investigate the effect of 2’-fucosyllactose (2’-FL) on the browning of white adipose tissue in 3T3-L1 adipocytes and C3H10T1/2 cells. The results revealed that 2’-FL decreased lipid accumulations with reduced intracellular triglyceride contents in vitro. 2’-FL intervention increased the mitochondria density and the proportion of UCP1-positive cells. The mRNA expressions of the mitochondrial biogenesis-related and browning markers (Cox7a, Cyto C, Tfam, Ucp1, Pgc1α, Prdm16, Cidea, Elovl3, Pparα, CD137, and Tmem26) were increased after 2’-FL intervention to some extent. Similarly, the protein expression of the browning markers, including UCP1, PGC1α, and PRDM16, was up-regulated in the 2’-FL group. Additionally, an adenosine monophosphate-activated protein kinase (AMPK) inhibitor, compound C (1 μM), significantly decreased the induction of thermogenic proteins expressions mediated by 2’-FL, indicating that the 2’-FL-enhanced beige cell formation was partially dependent on the AMPK pathway. In conclusion, 2’-FL effectively promoted the browning of white adipose in vitro.