The study aimed to evaluate the effects of L-carnitine on honey bee (Apis mellifera) drone spermatozoon at post-thaw. Semen samples obtained from sexually mature drones were pooled. Then, pooled semen was diluted with different concentrations of L-carnitine (2.5 mM/5 mM/10 mM) and without L-carnitine (control) supplemented extenders. Motility, plasma membrane functional integrity, acrosomal integrity and mitochondrial function were negatively affected by the cryopreservation process but DNA integrity was not affected. Malondialdehyde (MDA) concentration was used for analyzing the membrane lipid peroxidation status and a better result was obtained in the LC5 group compared with the control group at post-thaw. L-carnitine groups had a positive effect on sperm motility, plasma membrane integrity, and mitochondrial function compared to the control group (P<0.05). Acrosomal integrity was better preserved in the LC5 group compared to the control group. The study shows that LC supplemented extenders have beneficial effects on honey bee drone sperm parameters. The results of the present study demonstrated the beneficial effect of using a 5 mMol LC supplemented extender.