It is thought that mitochondrial dysfunction plays a role in the development of insulin resistance in type 2 diabetes. However, an overall unifying theory linking mitochondria with insulin action has yet to emerge, with many contradictions present in the literature. The aim of this study was to determine whether mitochondrial dysfunction is necessary and sufficient to induce cellular insulin resistance in 3T3L1 adipocytes and FAO hepatoma cells. We initially analysed multiple models of insulin resistance (glucose oxidase, tumor necrosis factor-α (TNFα) and chronic insulin in the adipose cell line and high glucose, TNFα and palmitate in the hepatocyte cell line) and comprehensively studied their effects on mitochondrial function, measuring basal mitochondrial respiration, ATP turnover, uncoupled respiration, membrane potential and superoxide production. Insulin resistance in these models, as assessed by insulin-stimulated 2-deoxyglucose uptake or insulin suppression of glucose production, could not be linked to a common defect in mitochondrial function. Indeed, TNFα had no effect on adipocyte mitochondrial function parameters, suggesting that mitochondrial dysfunction is not necessary for insulin resistance. To determine whether mitochondrial dysfunction is sufficient to induce insulin resistance, we treated cells with the mitochondrial inhibitors rotenone and oligomycin to induce site-specific mitochondrial dysfunction in both cell lines. Impairments in either complex I or ATP synthase induced insulin resistance in 3T3L1 adipocytes that was independent of mitochondrial ROS. In contrast, complex I inhibition in FAO hepatocytes enhanced insulin action, while inhibition of ATP synthase had no effect on insulin action. Site-specific impairments in mitochondrial metabolism were also associated with cell type-dependent alterations in mitochondrial function parameters and insulin signalling. These studies highlight the complex role of mitochondria in regulating insulin action, which cannot be accurately described by reductionist models, nor defined by universal statements.