Background: Tinospora tuberculata Bumee is a climbing vine distributed throughout the South Asian countries and it is known to be a hypoglycaemic agent. However, its role in cytoprotection and cellular oxidation is unclear. In the current investigation, cytoprotection against cellular stress and heat shock protein 70 (Hsp 70) modulation was investigated to understand the beneficial effect of butanolic fraction of T. tuberculata (TTB) in hyperglycemia and hyperlipidemia. Materials and Methods: The cytoprotective effect of TTB was studied on wild and knock-out yeast strain (Δtrx2). The effect of TTB on cell viability and the level of mitochondrial superoxide were determined to assess the antioxidant protection against reactive oxygen species assault. IR was induced in Wistar rats by high-fat diet and a low dose of streptozotocin. Hyperglycemic rats were orally treated with TTB (50 and 100 mg/kg body weight) or metformin (200 mg/kg b.w.) daily for 2 weeks. The effect of TTB on different parameters such as body weight, glucose levels, insulin resistance, glucose tolerance, lipid profile, antioxidant status, and hepatic Hsp 70 was analyzed in IR rats. TTB was characterized using high-resolution liquid chromatography-mass spectrometer (HR-LC/MS). Results: TTB demonstrated significant protection against the oxidative damage caused by H2O2 in both wild and Δtrx2 strains. In obese rats, TTB treatment reduced insulin resistance, improved glycaemic status, glucose tolerance, lipid profile, antioxidant level, and upregulated hepatic Hsp 70. HR-LC/MS analysis of TTB showed the presence of isoquinoline alkaloids (ambelline and reticuline), flavonoid glycoside (apiin), disaccharide carbohydrate (trehalose), cardenolide (calotropin), etc., Conclusion: The positive effect of the TTB on IR is attributed to its cytoprotective action against cellular stress and the activation of hepatic Hsp 70.