Glandular trichomes play a defensive role against herbivores in the leaves of many plant species. However, their functional role in inflorescences has not been studied, even though theory suggests that tissues with a higher fitness value, such as inflorescences, should be better defended. Using manipulative experiments, we analysed the defensive role of glandular trichomes against herbivorous insects in the inflorescence of Iberian columbines (genus Aquilegia), and its inter-population and inter-taxa variation in relation to herbivore abundance and potential selective pressure. The experiments were conducted in eight populations belonging to four subspecies of two columbines (Aquilegia vulgaris and Aquilegia pyrenaica). For each population, we estimated the density of glandular trichomes in the inflorescences, the abundance of insects stuck in the inflorescences, the abundance of small herbivorous insects, the incidence of damage on flowers and fruits, and the fruit set. The density of glandular trichomes on the inflorescence of A. vulgaris and A. pyrenaica was higher in regions of higher herbivore abundance. We also found that when the plants lose the protection of glandular trichomes, small insects have better access to flowers and fruits, causing more damage and reducing plant fitness. This study concludes that glandular trichomes are part of an adaptive response against phytophagous insect herbivory. The observed variation in herbivore pressure between taxa, likely caused by habitat differentiation, might have played a role in trait differentiation through divergent selection. This result adds evidence to the differentiation of the Iberian columbines through habitat specialization.