Abstract: This investigation aimed to identify the effects of neohesperidin on the differentiation of adipocyte and the underlying mechanism in 3T3-L1 preadipocytes. The cell viabilities of neohesperidin were detected in 3T3-L1 cells by MTS assay. The intracellular accumulation of lipids was visualized with Oil-red O staining and spectrophotometry analysis. The mRNA of CCAAT/enhancer binding protein α(C/EBPα) and peroxisome proliferators-activated receptorγ(PPARγ), adipogenic-specific genes during adipocyte differentiation, were measured by RT-PCR. Glycogen synthase kinase3β (GSK3β), glycogen synthase (GS) andprotein phosphorylation, the Akt signaling pathway, were analysed by immunoblotting. To confirm this GSK3β effect, 3T3-L1 cells were incubated with neohesperidin and GSK3β inhibitor (LiCl), and the intracellular accumulation of lipids and the level of adipogenic-specific protein were measured.The results showed that, neohesperidin significantly inhibited adipocyte differentiation and intracellular triglyceride formation (P<0.01), activated Akt signaling pathway, and promoted p-Akt and p-gsk3β, significantly inhibited C/EBPα, PPARγ, the expression of AP2 mRNA and protein (P<0.01). These effects were partly reversed by inhibition of GSK3β activity by LiCl. In summary, neohesperidin suppresses adipocyte differentiation via the Akt/GSK3β signaling pathway in 3T3-L1 preadipocytes.