Liquid storage of avian spermatozoa is currently being employed in programs utilizing the artificial insemination to optimize the management of genetically superior males. It is mandatory to use efficient semen storage techniques in order to prevent the reduction of the fertilizing ability of stored semen. The present study was designated to evaluate the effect of oleic acid on rooster semen quality stored at 4°C for 48 h. Semen was collected from 10 roosters twice a week. Good quality ejaculates were pooled and after dilution, the semen was enriched with 0 (control), 0.125 (O 0.125), 0.25 (O 0.25), 0.5 (O 0.5), and 1 (O1) millimolar oleate. Forward progressive motility and viability of spermatozoa were evaluated at 0, 24, and 48 h. Moreover, malondialdehyde (MDA) and total antioxidant activity (AOA) levels were measured in seminal plasma and spermatozoa at the mentioned time points. Motility was 80.33 ± 1.45, 80.00 ± 2.08, and 66.00 ± 2.30% at 24 h and 56.33 ± 1.45, 57.33 ± 2.18, and 41.33 ± 2.02% at 48 h in O 0.125, O 0.25, and control, respectively (P < 0.001). Total AOA concentrations of seminal plasma were significantly higher in oleate treated groups than the control at 24 and 48 h (P < 0.03). Moreover, concentrations of AOA in spermatozoa revealed that oleate treated group showed higher AOA values compared to the control group at 24 and 48 h (P < 0.001). MDA concentrations of seminal plasma and spermatozoa were lower in oleate treated groups in comparison with control group at 24 and 48 h (P < 0.05). In conclusion, rooster semen enrichment with low doses of oleate would exert beneficial effects on the quality of semen during cooled storage.