Semen cryopreservation technology plays a crucial role in enhancing animal reproductive efficiency and genetic resources preservation. However, the remarkable decline in fertility caused by sperm damage during the freezing process restricts its application in the poultry industry. The addition of antioxidants in semen is an effective approach to mitigate oxidative damage and enhance fertility. L-carnitine (LC) is an antioxidant and previous study indicated that it was related with sperm motility regulation. To explore its proper application in rooster semen cryopreservation, this study explored the effect of different levels of LC (0, 0.5 1.0, 2.5, 5.0, 7.5 mM) on post-tawed sperm motility, morphology, mitochondrial function, antioxidant activity, and fertility potential. The results demonstrated that sperm motility parameters including motility and motion parameters did not differ between groups. The sperm abnormality of 5.0 mM LC was lower than that in the control group (0 mM LC in the basic extender) (P <
0.05). The sperm plasma membrane integrity with 0.5, 2.5 and 5.0 mM LC was significantly higher than that in the control group (P <
0.05). All the treatment groups showed lower sperm reactive oxygen species than the control group. The 2.5 and 5.0 mM LC groups showed higher fertility (P <
0.05). Overall, this study provides empirical evidence supporting the effectiveness of LC for improving the efficiency of roosters' semen cryopreservation in a dose dependent manner, based on the results that supplementation of 2.5 and 5.0 mM LC in the basic extender improved the post-thawed sperm quality and fertility of roosters. In general, our study indicated that the addition of LC exhibited a notable impact on the freezing of rooster semen.