This study evaluated the possible protective role of chitosan nanoparticles (CNPs) on ram semen after cryopreservation using a Tris-egg yolk-based extender. Chitosan synthesis and characterization were done by transmission electron microscope (TEM), scanning electron microscope (SEM) and energy dispersive analysis of X-ray spectroscopy (EDAX). Semen ejaculates were collected by artificial vagina from five rams with proven fertility (two ejaculates / ram / week) for five weeks. Only good quality ejaculates were pooled and then diluted with tris extender supplemented with different concentrations of CNPs (0, 5, 15, 25, 50, and 75 μg/ml). The diluted Samples gradually cooled to 4°C over 90 min, then equilibrated for farther 2hrs in a cold Cabinet (4°C) after that packed in 0.5ml French straws, frozen on liquid nitrogen (LN) vapor for 15 minutes then stored in LN until evaluation.  Post-thawed samples were examined by computer-assisted sperm analysis (CASA) recording sperm motion parameters.  Additionally, the acrosome defects, membrane integrity, antioxidant activity as well as sperm ultrastructure, and DNA integrity were recorded. The results showed a significant improvement in total, rapid, and progressive motility, in addition to preserving the integrity of the acrosome, the plasma membrane, and the ultrastructure of sperm after adding (15 µg/ml) of chitosan nanoparticles. There was a non-significant increase in antioxidant activity with a decrease in the levels of lipid peroxides and no adverse effect on the DNA. It could be concluded that CNPs cross-linked with dextran sulfate were able to successfully protect the viability of frozen-thawed ram sperm by decreasing cryo-injury.