Sensing and resisting oxidative stress is critical for Vibrio cholerae to survive in either the aquatic environment or the gastrointestinal tract. Previous studies mainly focused on the mechanisms of oxidative stress response regulation that rely on enzymatic antioxidant systems, while functions of non-enzymatic antioxidants are rarely discussed in V. cholerae. For the first time, we investigated the role of hydrogen sulfide (H2S), the simplest thiol compound, in protecting V. cholerae against oxidative stress. We found that degradation of L-cysteine by putative cystathionine β-synthase (CBS) is the major source of endogenous H2S in V. cholerae. Our results indicate that intracellular H2S level has a positive correlation with cbs expression, while the enhanced H2S production can render V. cholerae cells less susceptible to H2O2 in vitro. Using proteome analysis and real-time qPCR assay, we found that cbs expression could stimulate the expression of several enzymatic antioxidants, including reactive oxygen species (ROS) detoxifying enzymes SodB, KatG and AhpC, the DNA protective protein DPS and the protein redox regulator Trx1. Assays of ROS detoxification capacities revealed that CBS-derived H2S could promote catalase activity at the post-translational level, especially for KatB, which serves as an important way that endogenous H2S participates in H2O2 detoxification. The enhancement of catalase activity by H2S is achieved through facilitating the uptake of iron. Adult mice experiments showed that cbs mutant has colonization defect, while either complementation of cbs or exogenous supplement of N-Acetyl-L-Cysteine restores its fitness in the host environment. Herein, we proposed that V. cholerae regulates CBS-dependent H2S production for better survival and proliferation under ROS stress.