The role of the recently-described interleukin-32 in Staphylococcus aureus-induced mastitis, an inflammation of the mammary gland, is unclear. We determined expression of IL-32, IL-6 and IL-8 in S. aureus- as compared to E.coli (Escherichia coli)-infected bovine mammary gland epithelial cells. Using live bacteria we found that in S. aureus-infected cells, induction of IL-6 and IL-8 expression was less pronounced than in E. coli-infected cells. Notably, IL-32 expression was decreased in S. aureus-infected cells, while it was increased in E. coli-infected cells. We identified the staphylococcal phenol-soluble modulin (PSM) peptides as key contributors to these effects, as IL-32, IL-6 and IL-8 expression by epithelial cells exposed to psm mutant strains was significantly increased as compared to cells exposed to the isogenic S. aureus wild-type strain, indicating that PSMs inhibit the production of these interleukins. The use of genetically complemented strains confirmed this observation. Inasmuch as the decreased expression of IL-32, which is involved in dendritic cell maturation, impairs immune responses, our results support a PSM-dependent mechanism that allows for the development of chronic S aureus-related mastitis.