The sperm DNA integrity post cryopreservation of human semen samples is one of the serious concerns in human infertility treatment. In the present study, the beneficial effects of zinc oxide nanoparticles in preserving the functional ability of spermatozoa was explored. Ejaculates of normozoospermic men cryopreserved along with Zinc oxide nanoparticles (ZnONPs) exhibited non-significantly higher percentage of total and progressive motility in frozen-thawed samples compared to control. The sperm chromatin damage and malondialdehyde (MDA) level was significantly lower in ZnONPs group (P < 0.01 and P < 0.05 respectively) and the spermatozoa's ability to undergo acrosome reaction was also unaltered. Fluorescence microscopy and High resolution transmission electron microscopy analysis demonstrated that the ZnONPs do not penetrate the membrane of spermatozoa but stay around the spermatozoa. In conclusion, the presence of ZnONPs during cryopreservation appears to be beneficial to the spermatozoa as they withstand freeze-thaw process competently better than control, without any adverse effect shown.