The aim of this study was to access the effect of HmsA, a 65-nt small regulatory RNA encoded by the pPCP1 plasmid, on virulence. Survival and the competition index were determined in mice infected with wild-type and an deletion mutant. RNA-seq was used to identify HmsA-regulated genes. HmsA deletion enhanced virulence. However, there was no overlap between 18 upregulated genes associated with pathogenicity and potential direct HmsA targets, based on gene expression screening after HmsA-pulse overexpression. HmsA inhibits virulence, but this effect may be mediated by indirect effects on pathogenesis, iron homeostasis and/or other cellular processes.