<p>Growing evidence indicates that host behaviors are regulated by the gut-brain interaction. In honeybees (<i>Apis mellifera</i>), a critical defensive behavior is the sting extension reflex. However, it remains unclear how the gut microbiota affects aversive learning and memory of this behavior. Here we demonstrate that the normal gut microbiota contributes to aversive learning and memory of the sting extension reflex in honeybees. Metabolomics analysis reveals that the gut microbiota affects tyrosine metabolism of the gut and hemolymph. Oral reintroduction of <i>Enterococcus faecium</i> or <i>Enterococcus faecalis</i> restores aversive learning and memory of microbiota-free honeybees, with an increase in their brain dopamine levels by supplying exogenous tyrosine. Conversely, applying fluphenazine, which decreases brain dopamine levels, weakens aversive learning and memory of honeybees in normal hives. These findings provide insights into the role of gut bacteria in regulating aversive learning and memory in honeybees, highlighting beneficial effects of the gut microbiota on the cognitive behaviors of social insects.</p>