The bacterium Vibrio cholerae colonizes the human small intestine and secretes cholera toxin (CT) to cause the rice-watery diarrhea characteristic of this illness. The ability of this pathogen to colonize the small bowel, express CT, and return to the aquatic environment is controlled by a complex network of regulatory proteins. Two global regulators that participate in this process are the histone-like nucleoid structuring protein (H-NS) and the general stress response regulator RpoS. In this study, we address the role of RpoS and H-NS in the coordinate regulation of motility and hemagglutinin (HA)/protease expression. In addition to initiating transcription of hapA encoding HA/protease, RpoS enhanced flrA and rpoN transcription to increase motility. In contrast, H-NS was found to bind to the flrA, rpoN, and hapA promoters and represses their expression. The strength of H-NS repression at the above-mentioned promoters was weaker for hapA, which exhibited the strongest RpoS dependency, suggesting that transcription initiation by RNA polymerase containing σ(S) could be more resistant to H-NS repression. Occupancy of the flrA and hapA promoters by H-NS was demonstrated by chromatin immunoprecipitation (ChIP). We show that the expression of RpoS in the stationary phase significantly diminished H-NS promoter occupancy. Furthermore, RpoS enhanced the transcription of integration host factor (IHF), which positively affected the expression of flrA and rpoN by diminishing the occupancy of H-NS at these promoters. Altogether, we propose a model for RpoS regulation of motility gene expression that involves (i) attenuation of H-NS repression by IHF and (ii) RpoS-dependent transcription initiation resistant to H-NS.