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This study intended to evaluate the probiotic potential of Lactobacillus fermentum RS-2 by in-vitro and in-vivo methods. Alloxan was injected intraperitoneally and animals were euthanised on 60th day post-challenge. The ameliorative effect of strain RS-2 alone and compare with standard strain ME-3, on glucose level, lipid parameters was performed. Oxidative stress was determined on the basis of enzyme markers. RS-2 strain showed a better survivability (55.60 ± 24%) in comparison with ME-3 (21.36 ± 27%) at pH 2.0 after 3 h and found potent antagonist against robust pathogens as compared to ME-3. The antioxidative activity of RS-2 cell lysate as% inhibition of linoleic acid peroxidation and % superoxide anion scavenging effect was observed to be 51.68 ± 0.32 and 43.5 ± 0.67%, respectively. Feeding rats with fermented milk containing RS-2 was found to significantly delay the severity of diabetes by amelioration of fasting blood glucose, and antioxidative stress markers in liver and blood as compared to diabetic control group.