1. Disruptive natural selection resulting from specialization on different hosts is recognized as one of the most important driving forces in the diversification of herbivores and parasites. It has been proposed that a similar mechanism could apply to carnivorous predators too, although the evidence is still lacking. 2. Here, we show that the differentiation of biotypes of specialized ant-eating spiders of the genus Zodarion has probably been induced by prey-shifting. We focused on two forms of one species Z. styliferum from the Iberian Peninsula that presumably represent ecological races. We conducted geographic, ecological, venom-oriented, reproductive and genetic divergence analysis among multiple populations collected at a number of sites across Portugal and Madeira. 3. Geographic analysis revealed that the two forms occur in mosaic sympatry. Each form was found to associate in nature with a different ant species in a different habitat. Specifically, the styliferum form hunted predominantly Messor ants, and the extraneum form hunted mainly Camponotus ants. Laboratory experiments revealed that the two forms exhibit a significant preference for attacking focal ants, demonstrating higher paralysis efficiency, and also show different venom composition. Cross-mating of the two forms was significantly less likely than between pairs of the same form, suggesting moderate assortative mating. Phylogenetic analyses indicate low genetic differentiation of the two forms and parallel-repeated evolution of biotypes. 4. Adaptive prey-shifting correlated with habitat preference are at present the most valid explanations for biotype formation in Zodarion. The speciation of ant-eating Zodarion spiders thus appears to follow a scenario similar to that of host-shifting in parasites and herbivores.