Optic Atrophy 1 (OPA1) is an inner mitochondrial membrane protein that plays important roles on inner membrane fusion, cristae remodeling and mitochondrial respiratory capacity. Although OPA1’s function has been determined in tissues such as the optic nerve and in skeletal muscle, the role of OPA1 in adipose tissue is incompletely understood. Our preliminary data demonstrated that conditional deletion of OPA1 in mature adipocytes during embryonic development prevented diet-induced obesity (DIO). We hypothesized this occurred due to a possible role for OPA1 on lipid metabolism and in adipogenesis during embryonic development. To circumvent possible developmental adaptations to OPA1 deletion, we developed mice in which OPA1 is selectively deleted in adipocytes in an inducible manner by crossing mice floxed for the OPA1 allele with mice harboring the tamoxifen inducible Cre-ERT2 under the control of the adiponectin promoter. At six weeks of age, mice were injected with tamoxifen for five days to induce the knockout of OPA1 in adipocytes. Immediately afterwards, these knockout mice (OPA1 Ad-Ind KO) were placed on a 60% high-fat diet (HFD). On a separate cohort fed regular chow, we confirmed OPA1 deletion in white (WAT) and brown (BAT) adipose tissue 4 weeks after tamoxifen injection. At this time, mice fed regular chow had similar body mass and body composition, as measured by nuclear magnetic resonance (NMR). Five weeks after being fed a HFD, we determined body composition by NMR, glucose homeostasis by glucose tolerance test and insulin sensitivity by insulin tolerance test. Our data demonstrated that OPA1 Ad-Ind KO mice had increased body mass, and exacerbated glucose intolerance and insulin resistance relative to littermate control mice, as demonstrated by impaired glucose and insulin tolerance tests, and elevated fasting glucose and insulin plasma levels. In conclusion, OPA1 deletion in adipose tissue of adult mice renders mice susceptible to DIO and insulin resistance. This was the opposite of what we observed when OPA1 was deleted during embryonic development, suggesting OPA1 plays a critical role in adipocyte development, while its absence in adult adipocytes impairs the ability of adipose tissue to adapt to caloric excess, resulting in exacerbated obesity.