Vibrio cholerae is the etiologic agent of the severe human diarrheal disease cholera. To colonize mammalian hosts, this pathogen must defend against host-derived toxic compounds, such as nitric oxide (NO) and NO-derived reactive nitrogen species (RNS). RNS can covalently add an NO group to a reactive cysteine thiol on target proteins, a process called protein S-nitrosylation, which may affect bacterial stress responses. To better understand how V. cholerae regulates nitrosative stress responses, we profiled V. cholerae protein S-nitrosylation during RNS exposure. We identified an S-nitrosylation of cysteine 235 of AphB, a LysR-family transcription regulator that activates the expression of tcpP, which activates downstream virulence genes. Previous studies show that AphB C235 is sensitive to O2 and reactive oxygen species (ROS). Under microaerobic conditions, AphB formed dimer and directly repressed transcription of hmpA, encoding a flavohemoglobin that is important for NO resistance of V. cholerae. We found that tight regulation of hmpA by AphB under low nitrosative stress was important for V. cholerae optimal growth. In the presence of NO, S-nitrosylation of AphB abolished AphB activity, therefore relieved hmpA expression. Indeed, non-modifiable aphBC235S mutants were sensitive to RNS in vitro and drastically reduced colonization of the RNS-rich mouse small intestine. Finally, AphB S-nitrosylation also decreased virulence gene expression via debilitation of tcpP activation, and this regulation was also important for V. cholerae RNS resistance in vitro and in the gut. These results suggest that the modulation of the activity of virulence gene activator AphB via NO-dependent protein S-nitrosylation is critical for V. cholerae RNS resistance and colonization.