Fla-CN (3-O-[(E)-4-(4-cyanophenyl)-2-oxobut-3-en-1-yl] kaempferol) is a semi-synthesized flavonoid derivative of tiliroside which exhibited anti-diabetic effect in vivo. Our previous study revealed the role of Fla-CN in anti-obesity and anti-diabetes in vivo, but the underlying mechanism remained to be addressed. The present study aimed to investigate the mechanism of anti-adipogenesis in vitro. Fla-CN markedly inhibited intracellular lipid accumulation in a dose-dependent manner, and the inhibitory effect was mainly limited to the early stage of adipocyte differentiation in vitro. Further investigations revealed that Fla-CN up-regulated the expression level of miR-27a/b and suppressed its target genes expression including peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα). Furthermore, the phosphorylation of AMP-activated protein kinase (AMPK) was also enhanced by Fla-CN in pre-adipocyte differentiation. These effects were abolished when cells were treated with miR-27a/b inhibitor and AMPK inhibitor Compound C. Additionally, Fla-CN reduced the expressions of adipocyte-specific genes such as sterol regulatory element-binding transcription factor 1c (SREBP-1c), fatty acid synthase (FAS) and adipocyte fatty acid binding protein (aP2). In conclusion, these results suggested a mechanism of Fla-CN for adipocyte differentiation inhibition of 3T3-L1 cells through miR-27a/b induction and AMPK activation.