© 2022The current study aimed to investigate the effects of rainbow trout seminal plasma (RTSP) on frozen-thawed honey bee drone spermatozoa. Semen samples were collected from sexually mature drones and pooled. Then pooled semen was diluted with different concentrations of RTSP (2.5%/5% / 10%) and without RTSP (control) supplemented extenders. Motility, plasma membrane functionality, acrosome integrity, and mitochondrial function were negatively affected by the cryopreservation process but DNA integrity was not affected. Membrane lipid peroxidation status was also analyzed using the malondialdehyde (MDA) concentration at frozen-thawed. RTSP10 groups had a positive effect on sperm motility (64.00 ± 3.38%), plasma membrane functionality (72.07 ± 2.12%), acrosome integrity (86.20 ± 2.11%), and mitochondrial function (69.33 ± 2.94%) compared to the control group (P< 0.05). The study shows that RTSP supplemented extenders have beneficial effects on at frozen-thawed drone sperm parameters. The results of the present study demonstrated the advantage of using a 10% RTSP supplemented extender for drone sperm motility, plasma membrane functionality, and mitochondrial function.