Background: Semen cryopreservation is a useful tool for preserving fertility for men involved in ART program who have been diagnosed with cancer and will undergo chemotherapy, radiotherapy or testicular surgery. Objective: The objective of this study to evaluate the effect of honey bee supplemented to cryoprotectant medium on post-thaw sperm DNA integrity ,morphology and round cells. Subjects ,Materials and Methods: Thirty semen samples were collected from 30 infertile patients. After assessment of semen analysis, semen samples were divided into 3 aliquots (0.7ml for each sample) and mixed with 1 ml of cryopreservation solution (G1,control) alone, enriched with 5% honey bee(G2) with 10% honey bee (G3) for cryopreservation. Current study used the acridine orange test to investigate the effect of honey bee in two concentration (5%, 10%) on the integrity of sperm chromatin structure pre-and post cryopreservation. Cryopreservation was done at -196 Co in liquid nitrogen and thawing was performed after six months. Direct swim up technique was used for in vitro sperm preparation post-thawing, sperm parameters were assessed and data were statistically analyzed pre-and post-thawing. Results: The results show there was a significant(P<0.05) increase in the percentage of morphologically normal sperm for all groups post-thawing, particularly for G3 group that was significantly (P<0.05) increased as compared to G1 and G2 groups, In contrast, non-significant differences (P>0.05) were observed between G1 and G2 groups. Sperm DNA fragmentation percentage (%) was significantly(P<0.05) increased in G1 post thawing without any additives as compared to pre-cryopreservation , where as in G2 and G3 sperm DNA fragmentation were increased but not significantly as post-cryopreservation groups. Also the results show that there were no significant differences between G2 and G3 whereas G3 gives better result as compared to G1 with significant differences. Round cells counts for all groups of post cryopreservation were significantly(P>0.05) decreased as compared to pre-cryopreservation. Non significant (P>0.05) differences in the round cells counts among all the groups of post -thawing in spite of G3 group has the lowest mean. Conclusions: From these results we concluded that supplementation of 10% of honey bee to the freezing cryoprotectant medium gives best protection to the morphology and DNA integrity of human sperm.