The effect of Honey bee supplementation into Tris extender on semen quality and bacterial activity of chilled- stored Barky ram' semen was studied. Semen samples were processed in Barky ram's semen extender with or without honey bee. Seven adult Barky rams aged 24-36 month and weighed 43.0± 1.5 kg were used. A total of 210 ejaculates were collected from the seven rams using an artificial vagina, 30 ejaculates each, twice weekly throughout the period of the study (July- October, 2016). After collection, each ejaculate was diluted (1:10) with Tris–based extender at 37˚C immediately after dilution, the specimens were split into 4 aliquots; the first aliquot served as control (C), whereas the other 3 aliquots were supplemented with 1.5 (HB 1.5), 3 (HB 3) and 4.5 (HB 4.5) ml honey bee/100 ml extender. Both control and treated specimens were subjected to chilled preservation at 5˚C for a period of 48 h, during which semen physical characteristics were evaluated, immediately after dilution time and every 24 h thereafter until 48 h storage at 5˚C.  Microbial contamination in all specimens was also determined at 0, 24 and 48 h. The results showed that supplementing semen extender with honey bee improved (P<0.05) sperm motility and reduced   (P < 0.05) percent of dead and abnormal spermatozoa and acrosomal damage. In conclusion, supplementing semen extender with honey bee for ram semen preservation at 5°C has reduced the deleterious effects due to chilling condition. This would facilitate the application of assisted reproductive technologies; i.e. artificial insemination and in vitro fertilization in sheep by extending the storagability and survival of sheep sperm.