The Type VI Secretion System (T6SS) is used by bacteria for virulence, resistance to grazing and competition with other bacteria. We previously demonstrated that the role of the T6SS in interbacterial competition and in resistance to grazing is enhanced in Vibrio cholerae in the presence of subinhibitory concentrations of polymyxin B. Here, we performed a global quantitative proteomic analysis and a targeted transcriptomic analysis of the T6SS known regulators in V. cholerae grown with and without polymyxin B. The proteome of V. cholerae is greatly modified by polymyxin B with more than 39 % of the identified cellular proteins displaying a difference in their abundance, including T6SS-related proteins. We identified a regulator whose abundance and expression are increased in the presence of polymyxin B, vxrB, the response regulator of the two-component system VxrAB (VCA0565-66). In vxrAB, vxrA and vxrB deficient mutants, the expression of both hcp copies (VC1415 and VCA0017), encoding an identical protein Hcp which is the major component of the T6SS syringe, although globally reduced, was not modified by polymyxin B. Thus, the upregulation of the T6SS in the presence of polymyxin B appears to be, at least in part, due to the two-component system VxrAB.