Background: Sperm preservation at a cool temperature reduces sperm metabolism while preserving its viability and reproductive ability. Researchers have sought to extend semen preservation effectiveness for more than 24 hours. Due to the particular physiological characteristics of small ruminant spermatozoa, the cooling procedure decreases its reproductive ability. Objectives: This study aimed to determine the effect of adding L-carnitine (LC) to the cooling extender on the quality of the ram’s sperm following cooling preservation at 4°C.Methods: The collected sperm samples were diluted and divided into 4 groups with varying doses of LC supplementation (0, 1, 5, and 10 mM). The samples were kept at 4°C for up to 48 hours. At 0, 24, and 48 hours of cooling, the sperms’ total motility, progressive motility, viability, lipid peroxidation, membrane integrity, and mitochondrial activity were assessed. Results: The results showed that different treatments did not affect the quality of semen samples at time 0 of cooling storage (P>0.05). Cooling medium supplemented with 5 mM LC demonstrated improved total motility, progressive motility, viability, membrane integrity, and mitochondrial activity compared to the other groups after 24 and 48 hours of cooling (P≤0.05). Furthermore, after 24 and 48 hours of storage, 5 mM LC produced less lipid peroxidation (P≤0.05) than the other treatments.Conclusion: In conclusion, reinforcing ram’s cooling storage medium with 5 mM LC protects ram semen samples against cold-induced structural and functional impairment throughout 24- and 48-h storage.