A honeybee that is away from the hive foraging for nectar or pollen will rarely sting, except when stepped on or roughly handled. Honeybees will actively seek out and sting when they perceive the hive to be threatened, often being alerted to this by the release of attack pheromones.

Although it is widely believed that a worker honeybee can sting only once, this is a misconception: although the stinger is in fact barbed so that it lodges in the victim's skin, tearing loose from the bee's abdomen and leading to its death in minutes, this only happens if the victim is a mammal (or bird). The bee's stinger evolved originally for inter-bee combat between members of different hives, and the barbs evolved later as an anti-mammal defense: a barbed stinger can still penetrate the chitinous plates of another bee's exoskeleton and retract safely. Honeybees are the only hymenoptera with a barbed stinger.

The stinger's injection of apitoxin into the victim is accompanied by the release of alarm pheromones, a process which is accelerated if the bee is fatally injured. Release of alarm pheromones near a hive or swarm may attract other bees to the location, where they will likewise exhibit defensive behaviors until there is no longer a threat (typically because the victim has either fled or been killed). These pheromones do not dissipate nor wash off quickly, and if their target enters water, bees will resume their attack as soon as the target leaves.