Workers never mate in the large majority of ants, and they have usually lost the spermatheca, an organ specialized for long-term storage of sperm. Such 'non-sexual' workers are restricted to laying unfertilized eggs that give rise to males, and they cannot compete with the queens for the production of female offspring. In sharp contrast, workers in 200-300 species from phylogenetically basal subfamilies can reproduce sexually ('gamergates') because they retain a functional spermatheca like the queens. Importantly, 'non-sexual' workers in closely related species have a vestigial spermatheca. In this study, we compared the reservoir epithelium of 'sexual' workers to that of congeneric queens and 'non-sexual' workers using 21 species of Amblyoponinae, Ponerinae and Ectatomminae. We show that a pronounced thickening of the epithelium near the opening of the sperm duct is strictly associated with sexual reproduction in both castes. This is unlike 'non-sexual' workers in which this epithelium is always very thin, with few organelles; but all other structures remain intact. We discuss this evolutionary degeneration of the spermatheca and how it relates to behavioural or physiological modifications linked to mating. Our results help understand the loss of sexual reproduction by ant workers, a critical step in the extreme specialization of their phenotype.