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The present study compared the effect of mitochondria-targeted (Mitoquinone, MitoQ) and untargeted cytosolic antioxidant (Resveratrol, RESV) supplementation on lipid peroxidation (LPO) and in-vitro sperm functions of cryopreserved buffalo bull semen. To optimize additive's concentration, sperm pellet obtained from twenty-four ejaculates was supplemented with different concentrations of MitoQ (20 nM, 100 nM, 200 nM); and RESV (10 μM, 25 μM, 50 μM) against control in the extender. The post-thaw sperm motility, livability, and membrane integrity were higher (P 0.05) in 200 nM MitoQ and 50 μM RESV than other concentrations used. In another experiment, sperm pellet from thirty-two ejaculates was supplemented with 200 nM MitoQ and 50 μM RESV in the extender. Pre-freeze and post-thaw progressive motility and livability were higher (P 0.05) in MitoQ (200 nM) than RESV (50 μM) treatment. MitoQ supplementation improved post-thaw membrane integrity (CFDA-PI) higher (P 0.05) than RESV, however, hypo-osmotic swelling response observed no improvement with RESV treatment. Post-thaw LPO rate was lower (P 0.05) and Bovine cervical mucus penetration was higher (P 0.05) in MitoQ than RESV treatment. In post-thaw semen, MitoQ showed higher (P 0.05) proportion of acrosome intact (FITC-PNA), live non-apoptotic (P 0.01) sperm with a higher reduction (P 0.05) in membrane scrambling. MitoQ improved (P 0.01) proportion of sperm with high Mitochondrial Membrane Potential and low LPO (P 0.01) than RESV treatment. In conclusion, improvement in post-thaw in-vitro sperm functions and cryo-tolerance was more evident in MitoQ than RESV supplemented buffalo bull semen. Our study provides a better strategy to mitigate oxidative stress by enhancing mitochondrial antioxidant system with targeted antioxidants than cytosolic antioxidant supplementation.