Emerging evidence suggests that environmental pollutants, including insecticides, are linked to excessive weight gain and altered glucose homeostasis. However, there is currently limited knowledge regarding the biochemical mechanisms by which exposure to insecticides contribute to obesity and its associated pathologies. We first studied the influence of fipronil (a phenylpyrazole family insecticide) on 3T3-L1 adipogenesis. Recently imidacloprid, a neonicotinoid insecticide, was reported to potentiate adipogenesis and insulin resistance in vitro. The current studies also determined the effects of imidacloprid exposure and its interaction with dietary fat in the development of adiposity and insulin resistance using both male and female C57BL/6J mice. Current results suggest that fipronil and imidacloprid alter adipogenesis and results in increased lipid accumulation via a AMPKα-mediated pathway in cell culture models. The current results also suggest that imidacloprid significantly promoted high fat diet-induced body weight gain and adiposity in mice (both males and females). In addition, imidacloprid treatment with high fat diet resulted in impaired glucose metabolism in male C57BL/6J mice. Consistently, there were significant effects of imidacloprid on key regulators of lipid and glucose metabolisms, including AMP-activated protein kinase-α (AMPKα) in the white adipose tissue and the liver.