Mitochondria are subjected to direct attack by large amounts of reactive oxygen species and compromised mitochondrial function ultimately causes neuronal apoptosis and neurodegeneration causing cognitive dysfunctions and neuropathy. Therefore, therapies based on maintaining mitochondrial integrity might be highly beneficial in such conditions. The motive behind present study is to investigate the outcome of genistein treatment against mitochondrial function impairment in brain of streptozotocin-induced diabetic mice. The mitochondrial suspension from cortex of diabetic mice was evaluated for any dysfunction aligned with levels of mitochondrial respiratory chain enzymes, thiols and oxidative stress. Mitochondrial apoptosis has been examined by investigating caspase-3 activation and cell viability by MTT assay. Chronic genistein treatment (2.5, 5.0 and 10.0 mg/kg, i.p., twice daily) significantly restored levels of mitochondrial respiratory chain enzymes (complex I, II, III and IV), reversed oxidative stress (malondialdehyde and superoxide dismutase levels), increased mitochondrial thiols (protein, non-protein and total thiols), decreased neuronal apoptosis (caspase-3 activity) and increased cellular viability (MTT assay) in the mitochondrial suspension from the cortex of streptozotocin-induced diabetic mice. Treatment with metformin also improved diabetes induced mitochondrial function impairment and significantly dragged these biochemical parameters towards respective controls. The study signifies that genistein supplementation might be a prospective therapeutic approach for the treatment and/or avoidance of diabetic linked neuronal dysfunction.