This study was carried out to evaluate the efficiency of supplementing ram semen diluent with glutathione (GSH) on liquid-chilled and cryo-storage capacities of spermatozoa. Twenty ejaculates were collected from 5 Barki rams, 4 ejaculates each, during late-January and mid-February using an artificial vagina. The pooled specimens of each collection session were diluted (1:10) with Tris-citric egg yolk extender, and were split into 5 aliquots. The first aliquot served as control (GSH-free), whereas the other 4 aliquots were supplemented with 0.1, 0.2, 0.3 or 0.4 mMGSH. Thereafter, the samples were stored at 4°C for 3 h whereat each sample was subdivided into two portions. The first portion was maintained at 4°C for the subsequent 48 h during which sperm traits were assessed alongside oxidative stress indicators, whereas a chilled-glycerolized base diluent was added to the other portion and was further processed for cryopreservation. Post-thaw physical and kinematic properties of spermatozoa were evaluated by a computer-assisted sperm analysis (CASA) system, and sperm DNA fragmentation level within each group was also determined by fluorescent imaging. The results showed that GSH supplementation reduced (P<0.05) the oxidative stress on spermatozoa and maintained their physical and morphological criteria over the 48h period of chilled storage in a dose-depending pattern. Moreover, the 0.4 mM level of GSH improved (P<0.05) post-thaw traits and kinematics of ram sperm while decreasing (P<0.05) DNA fragmentation level compared to the control specimens. These findings imply glutathione's powerful potential as an exogenous antioxidant supplement in ram sperm medium which could be of great significance for further application in semen processing and IVF workflows