The inhibition of adipocyte differentiation has a significant role on the prevention of obesity and obesity-associated complications.In this study, we aimed to detect whether hyperoside is able to inhibit the conversion of pre-adiposits into mature adiposits.3T3-L1 pre-adipocytes were stimulated so as to differentiate into mature adipocytes. Hyperoside in non-cytotoxic concentrations (1, 2, 5, and 10 µM) were separately applied to differentiated 3T3-L1 cells. Oil red O staining was performed and triacylglycerol contents were measured. Furthermore, gene and protein expressions of transcription factors, adipogenic genes and adipokines were examined in order to investigate the effect of hyperoside on adipocyte differentiation.Hyperoside in high concentrations significantly suppressed the adipogenic process by inhibiting the expression of transcription factors and adipogenic genes and reducing lipid accumulation in adipocytes (p<0.05). Low doses of hyperoside are able to inhibit adipogenesis, but higher doses are needed to reduce fat accumulation in mature adipocytes. In the case of maturing preadipocytes, 5 μM of hyperoside exerts its antiadipogenic effect at the early stages of adipogenesis, whereas 10 μM of hyperoside acts at the later stages (p<0.05).These results suggest that hyperoside has a beneficial effect on the prevention and treatment of obesity.