Introduction Uptake of Hexavalent chromium (Cr 6) by the testis and its subsequent reduction to trivalent chromium is well known.Several studies attributed Cr 6 testicular toxicity to oxidative stress. Vitamin E is a lipid soluble antioxidant prevents damage to membranes. Selenium (Se) is an essential micronutrient with an antioxidant activity. This study aimed to investigate the potential protective role of vitamin E and selenium against sub-chronic toxicity of Cr 6 on testes of adult male albino rats through biochemical, cytogenetic and sperm analysis. Methodology: The duration of the present study was six weeks and was carried out on 60 adult male albino rats. They were divided into 10 rats of six groups, control group, selenium group (0.5 mg/kg by oral gavage), vitamin E group (125 mg/kg by oral gavage), Cr 6 group (10 mg/kg by oral gavage), Cr 6 + Selenium group and Cr 6 + Vitamin E group. Biochemical, cytogenetic and sperm analysis were done to all groups. Results: Levels of cholesterol, triglyceride, micronucleus polychromatic erythrocyte, total sperm abnormality are increased while glucose, serum glutathione-s-transferase, testosterone, DNA and RNA contents, sperm viability and content decreased. Treatment with Se or vit.E improved all these effects. Conclusion: Hexavalent chromium induced changes in biochemical, cytogenetic and sperm analysis, which lead to disruption in reproductive cell functions. Se or vit. E has the ability for reduction of these deleterious effects. Recommendation: Since vitamin E and selenium have antioxidant properties and play vital role in preventing hexavalent chromium induced toxicity, dietary supplementation with them isrecommended for exposed workers and general population.