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As one of the most successful pathogenic organisms, Vibrio cholerae (V. cholerae) has evolved sophisticated regulatory mechanisms to overcome host stress. During long-term colonization by V. cholerae in adult mice, many spontaneous nonmotile mutants (approximately 10% at the fifth day post-infection) were identified. These mutations occurred primarily in conserved regions of the flagellar regulator genes flrA, flrC, and rpoN, as shown by Sanger and next-generation sequencing, and significantly increased fitness during colonization in adult mice. Intriguingly, instead of key genes in DNA repair systems (mutS, nfo, xthA, uvrA) or ROS and RNS scavenging systems (katG, prxA, hmpA), which are generally thought to be associated with bacterial mutagenesis, we found that deletion of the cyclin gene dps significantly increased the mutation rate (up to 53% at the fifth day post-infection) in V. cholerae. We further determined that the dpsD65A and dpsF46E point mutants showed a similar mutagenesis profile as the Δdps mutant during long-term colonization in mice, which strongly indicated that the antioxidative function of Dps directly contributes to the development of V. cholerae nonmotile mutants. Methionine metabolism pathway may be one of the mechanism for ΔflrA, ΔflrC and ΔrpoN mutant increased colonization in adult mice. Our results revealed a new phenotype in which V. cholerae fitness increases in the host gut via spontaneous production nonmotile mutants regulated by cyclin Dps, which may represent a novel adaptation strategy for directed evolution of pathogens in the host. Author summary: Pathogen–host coevolution during long-term infection is the main strategy by which pathogens adapt to the host environment and coexist with humans. The host usually initiates a series of immune responses against the pathogen by sensing pathogen-associated molecular patterns, including flagella and lipopolysaccharide. Among them, the reactive oxygen/nitrogen species (ROS/RNS) produced by the host can quickly lead to loss of the cytoplasmic membrane and DNA damage in pathogens, and directly kill the pathogens or inhibit their growth. V. cholerae, one of the oldest pathogenic enteric bacteria, has evolved elaborate regulatory mechanisms in response to ROS, including inhibition of ROS production by Dps and scavenging of ROS by PrxA, KatG and KatB. Here, we report a novel adaptation strategy in which V. cholerae produces high-frequency spontaneous nonmotile mutants during long-term colonization in the adult mouse gastrointestinal tract. In the nonmotile mutants, we identified that the hotspots for the spontaneous mutations are the flagellar regulatory genes flrA, flrC and rpoN. These nonmotile mutants exhibited enhanced host adaptation in adult mice, possibly through distinct mechanisms. Further studies revealed that only cyclin Dps, not the classic DNA repair systems or ROS and RNS scavenging systems, were closely involved in mutagenesis in the nonmotile mutants. These results may represent a novel adaptation strategy for the directed evolution of pathogens in the host. Our results might explain why many clinical isolates are nonmotile in cholera outbreak areas such as Haiti and Nepal. The spontaneously generated nonmotile mutants of V. cholerae during long-term colonization exhibited increased adaptation in the host and the potential to become epidemic strains, which warrants the attention of epidemiologists and bacteriologists. [ABSTRACT FROM AUTHOR]