Ectoparasites are ubiquitous and are often harmful to host fitness. Whereas protective responses to ectoparasitism in vertebrate hosts are well documented, our understanding of such defences in invertebrates remains limited. Here, we examined attachment-resistance in adult Drosophila to their naturally co-occurring ectoparasitic mites, Gamasodes pachysetis (Parasitidae). Significant differences in mite attachment duration were documented among 6 species of Drosophila, providing evidence for interspecific differentiation in attachment-resistance. Experiments with D. malerkotliana, a species exhibiting a relatively high rate of mite detachment, revealed that pre-infesting flies significantly reduced mite attachment duration compared to naïve controls, indicating a priming effect. In contrast, a reduction in attachment duration was not observed in D. malerkotliana after experimentally wounding the abdominal cuticle. These results suggest that the priming effect is not simply a response to cuticle damage, and that its activation may depend on mite-specific factors. Eight genes were individually tested for their effects on the rate of mite detachment from adult flies by deploying the GAL4-UAS gene knockdown system in D. melanogaster. Knockdown of heat shock protein 70Ba (Hsp70Ba) and prophenoloxidase 2 (PPO2), which underlie general stress and melanization responses, respectively, significantly prolonged mite attachment duration, implicating their involvement in host attachment-resistance to mites. Together the results support the existence of inducible protective mechanisms mediating parasitism by mites in a naturally occurring invertebrate host–ectoparasite symbiosis.