Honey bees (Apis mellifera) (Hymenoptera: Apidae) are social insects that have evolved a coordinated defensive response to ensure colony survival. Their nests may contain valuable resources such as pollen and nectar that are attractive to a range of insect and mammalian intruders and need protecting. With sufficient provocation, honey bees will mobilize and sting intruders, who are likely to incur additional stings. To inspect and manage their colonies, beekeepers apply smoke to decrease the likelihood of being stung. The use of smoke is a ubiquitous beekeeping practice, but the reasons behind its efficacy remain unknown. In this study, we examined the effects of smoke on honey bee defensive behavior by assessing individual sting extension responses under smoke conditions. We applied a brief voltage to the bee, ranging from a mild to a strong perturbation, and assessed four components of the sting extension reflex using two types of smoke. We found that smoke did not influence the probability of sting extension, but it did affect whether a venom droplet was released with the stinger. The venom droplet was more likely to be released at higher voltage levels, but this effect was significantly reduced under smoke conditions. Based on these results, we propose that the venom droplet coincides with greater agitation in individual bees; and smoke reduces the probability of its release. We speculate that the venom droplet serves to amplify the sting alarm pheromone, and smoke, in its ability to reduce droplet formation, may indicate that less alarm pheromone is released.