Excessive weight and obesity are associated with the development of diabetes mellitus type 2 (DMII) in humans. They also pose high risks of Staphylococcus aureus colonization and overt infections. S. aureus causes a wide range of severe illnesses in both healthy and immunocompromised individuals. Among S. aureus virulence factors, superantigens are essential for pathogenicity. In this study, we show that rabbits that are chronically exposed to S. aureus superantigen toxic shock syndrome toxin-1 (TSST-1) experience impaired glucose tolerance, systemic inflammation, and elevated endotoxin levels in the bloodstream, all of which are common findings in DMII. Additionally, such DMII-associated findings are also seen through effects of TSST-1 on isolated adipocytes. Collectively, our findings suggest that chronic exposure to S. aureus superantigens facilitates the development of DMII, which may lead to therapeutic targeting of S. aureus and its superantigens. IMPORTANCE: Obesity has a strong correlation with type 2 diabetes, in which fatty tissue, containing adipocytes, contributes to the development of the illness through altered metabolism and chronic inflammation. The human microbiome changes in persons with obesity and type 2 diabetes, including increases in Staphylococcus aureus colonization and overt infections. While the microbiome is essential for human wellness, there is little understanding of the role of microbes in obesity or the development of diabetes. Here, we demonstrate that the S. aureus superantigen toxic shock syndrome toxin-1 (TSST-1), an essential exotoxin in pathogenesis, induces inflammation, lipolysis, and insulin resistance in adipocytes both in vitro and in vivo. Chronic stimulation of rabbits with TSST-1 results in impaired systemic glucose tolerance, the hallmark finding in type 2 diabetes in humans, suggesting a role of S. aureus and its superantigens in the progression to type 2 diabetes.