There is emerging evidence that organochlorine and organophosphorus insecticide exposure may be linked to excessive weight gain and symptoms of diabetes. However, there is a lack of knowledge for other types of insecticides with potential influence on obesity and diabetes. Thus, the purpose of this investigation was to determine the role of imidacloprid, a neonicotinoid insecticide, in lipid metabolism by use of 3T3-L1 adipocytes. Imidacloprid treatment potentiated lipid accumulation in 3T3-L1 adipocytes and significantly increased expression of a key regulator of adipocyte differentiation and key regulators of lipogenesis. These results imply the involvement of imidacloprid in altered adipogenesis, resulting in increased fat accumulation. This finding is the first report of a potential link between neonicotinoid insecticide exposure and lipid accumulation in adipocytes. Further in vivo as well as epidemiological studies will be required before we can extrapolate these findings to a potential contribution of imidacloprid in human obesity.