ABSTRACT Listeria monocytogenes σ ^B positively regulates the transcription of class II stress response genes; CtsR negatively regulates class III stress response genes. To identify interactions between these two stress response systems, we constructed L. monocytogenes Δ ctsR and Δ ctsR Δ sigB strains, as well as a Δ ctsR strain expressing ctsR in trans under the control of an IPTG (isopropyl-β- d -thiogalactopyranoside)-inducible promoter. These strains, along with a parent and a Δ sigB strain, were assayed for motility, heat resistance, and invasion of human intestinal epithelial cells, as well as by whole-genome transcriptomic and quantitative real-time PCR analyses. Both Δ ctsR and Δ ctsR Δ sigB strains had significantly higher thermotolerances than the parent strain; however, full heat sensitivity was restored to the Δ ctsR strain when ctsR was expressed in trans . Although log-phase Δ ctsR was not reduced in its ability to infect human intestinal cells, the Δ ctsR Δ sigB strain showed significantly lower invasion efficiency than either the parent strain or the Δ sigB strain, indicating that interactions between CtsR and σ ^B contribute to invasiveness. Statistical analyses also confirmed interactions between the ctsR and the sigB null mutations in both heat resistance and invasion phenotypes. Microarray transcriptomic analyses and promoter searches identified (i) 42 CtsR-repressed genes, (ii) 22 genes with lower transcript levels in the Δ ctsR strain, and (iii) at least 40 genes coregulated by both CtsR and σ ^B , including genes encoding proteins with confirmed or plausible roles in virulence and stress response. Our data demonstrate that interactions between CtsR and σ ^B play an important role in L. monocytogenes stress resistance and virulence.