OPA1 (Optic Atrophy 1) is a mitochondrial GTPase that regulates mitochondrial dynamics, cristae structure and respiratory capacity. Changes in mitochondrial dynamics can alter brown adipocyte metabolism in vitro and impair thermogenic response in vivo. Additionally, OPA1 has been proposed to mediate adrenergic control of lipolysis in adipocytes. However, the specific contribution of OPA1 to adipose tissue physiology in vivo is not known. OPA1 floxed mice were crossed with mice harboring the Cre recombinase under the control of the adiponectin promoter, for specific deletion of the Opa1 gene in adipocytes (OPA1Adipo-/-). Under basal conditions, young (10 weeks of age) OPA1Adipo-/- mice had similar body mass compared to wild type (WT) mice, however BAT was enlarged and resembled white adipose tissue (WAT) histologically. By electron microscopy, OPA1Adipo-/- mice exhibited smaller mitochondria with deranged cristae structure. In addition, mitochondrial complexes I and III protein levels were reduced in BAT and WAT of OPA1Adipo-/- mice, suggesting impaired mitochondrial oxidative capacity. Following weaning mice were placed on a control diet (10% fat) or a high-fat diet (60% fat) for 12 weeks. Surprisingly, OPA1 deletion completely prevented diet-induced weight gain at the expense of reduced total fat mass and decreased individual fat pad weights. OPA1Adipo-/- had reduced liver mass and circulating free fatty acids (FFA) when fed a HFD compared to WT mice. Energy expenditure was increased in OPA1Adipo-/-, despite no significant changes in food intake and activity levels. Moreover, diet-induced insulin resistance was also ameliorated in OPA1Adipo-/- mice, as shown by improved glucose and insulin tolerance and reduced serum insulin levels. FGF21 circulating levels were elevated in OPA1Adipo-/- mice, which correlated with increased FGF21 protein levels in WAT and BAT. Taken together, these data identify a fundamental role for OPA1 in lipid metabolism, with its absence completely preventing fat mass expansion in response to a HFD and improving insulin sensitivity, by mechanisms that may involve induction of FGF21.