Abstract: The aims were to study the pollination characteristics of honeybee in cotton CMS lines, and whether honeybee was suitable for production of three-line hybrid cotton or not. In this experiment, honeybee (Apis mellifera Ligustica Spinola) was used as pollinators on cotton cytoplasmic male sterile (CMS) lines in net house. The artificial pollination was used as a control. We investigated the visit frequency and single flower retention time of honeybee on cotton CMS maintainer and CMS lines in fine and cloudy days, and compared the agronomic traits, the yield of seed and the cost. In fine days, the visiting frequency of honeybee in cotton CMS maintainer and CMS lines were 60 and 49.2 times per hour, respectively, and the difference was significant. In cloudy days, the visiting frequency of honeybee in the two type lines were 23.4 and 13.8 times per hour, respectively, and the difference was not significant. In comparison of the characters between honeybee and artificial pollination, the difference of seed number per boll was significant, while boll number and empty branches per plant had extremely significant difference. The number of fruit branches, boll weight, lint percentage, seed index, germination rate had no significant difference. The hybrid seed production of bee pollination could reach about 70% of artificial pollination. But its cost was two-third of artificial pollination. Therefore, the rational use of honeybee pollination technology could play an important role in cotton CMS hybrid seed production.