The acquisition of new genes, via horizontal transfer or gene duplication/ diversification, has been the dominant mechanism thus far implicated in the evolution of microbial pathogenicity. In contrast, the role of many other modes of evolution—such as changes in gene expression regulation—remains unknown. A transition to a pathogenic lifestyle has recently taken place in some lineages of the budding yeast Saccharomyces cerevisiae. Here we identify a module of physically interacting proteins involved in endocytosis that has experienced selective sweeps for multiple cis-regulatory mutations that down-regulate gene expression levels in a pathogenic yeast. Genetic variants at these loci are associated with pathogenicity across 88 diverse yeast strains, suggesting the adaptations may have increased virulence. To test this, we created a panel of single-allele knockout strains whose hemizygous state mimics the genes’ adaptive down-regulations, and measured their virulence in a mammalian host. Despite having no growth advantage in standard laboratory conditions, nearly all of the strains were more virulent than their wild-type progenitor, suggesting that these adaptations likely played a role in the evolution of pathogenicity. These results suggest that cis-regulatory adaptation can occur at the level of physically interacting modules, and that one such polygenic adaptation led to increased virulence during the evolution of a pathogenic yeast.