Leptospira interrogans , the causative agent of most cases of human leptospirosis, must respond to myriad environmental signals during its free-living and pathogenic lifestyles. Previously, we compared L. interrogans cultivated in vitro and in vivo using a dialysis membrane chamber (DMC) peritoneal implant model. From these studies emerged 160 genes that were differentially regulated in response to host signals, including perRA , one of two Peroxide stress response (PerR)-like regulators encoded by L. interrogans . Zavala-Alvarado et al . recently demonstrated that leptospires lacking both PerRA and PerRB are avirulent in hamsters. Herein, we establish that PerRA and PerRB also are required for renal colonization in C3H/HeJ mice. The finding that loss of virulence was observed only with the perRA/B double mutant suggests that these regulators serve redundant or overlapping functions in vivo . Our finding that the perRA / B double mutant survives at wild-type levels in DMCs is noteworthy as it demonstrates that the loss of virulence is not due to a metabolic lesion ( i.e. , metal starvation) but instead reflects dysregulation of virulence-related gene products. Comparative RNA-seq analyses of perRA , perRB and perRA/B mutants cultivated within DMCs identified 106 genes that are dysregulated only in the double mutant, including ligA, ligB and lvrA/B sensory histidine kinases. Decreased expression of LigA and LigB in the perRA / B mutant was not due to loss of LvrAB signal transduction. The majority of genes in the PerRA and PerRB DMC regulons was differentially expressed only in vivo , highlighting the importance of host-specific signals for regulating gene expression in L. interrogans . Importantly, the PerRA, PerRB and PerRA/B DMC regulons each contain multiple genes related to environmental sensing and/or transcriptional regulation. Collectively, our data suggest that PerRA and PerRB are part of a complex signaling network required by L. interrogans for adaptation to and survival within the host.