Epidemiological studies have revealed a correlation between a long-term exposure to organochlorine pesticides (OCPs) and type 2 diabetes mellitus (T2DM). However, supportive evidence from pathological studies in rodent models remains limited. In this study, we addressed this gap by investigating the metabolic effects of a defined OCP mixture (OCPM)-hexachlorobenzene, β-hexachlorocyclohexane, p,p՛-DDT, heptachlor, and chlordane (1:1:1:1:1) was administered orally to C57BL/6J mice at doses of 0.5 mg/kg and 2.0 mg/kg of body weight (BW) for 90 days. This OCPM reflects environmentally relevant co-exposure patterns that have rarely been examined in animal studies to date. Unlike prior studies, this study investigates the chronic effects of a low-dose, mixture toxicity of OCPs in a controlled in vivo model, providing novel insights into the metabolic disruption caused by chemical mixtures. Chronic low-dose exposure to OCPM significantly impaired glucose homeostasis, evidenced by elevated fasting blood glucose, plasma glucose, insulin and free fatty acid, along with increased HOMA-IR and oral glucose intolerance, which are associated with insulin resistance. At molecular level, OCPM significantly altered multiple metabolic pathways, including insulin signaling, mitochondrial function, oxidative stress response, gluconeogenesis, and lipid metabolism, in both skeletal muscle and liver tissues. These changes are all associated with the development of T2DM. Notably, the effects were more pronounced at lower dose of 0.5 mg/kg-BW than the higher dose, suggesting possible non-linear response. Collectively, these findings underscore the diabetogenic potential of chronic low-dose exposure of OCPM and provide a novel insight into how persistent environmental mixtures may contribute to the onset and progression of insulin resistance and T2DM.