Abstract Background Enterobacter strains can display heterogeneous resistance (heteroresistance) to colistin but the mechanisms remain largely unknown. We investigated potential mechanisms of colistin heteroresistance in an Enterobacter clinical strain, WCHECl-1060, and found a new mechanism. Methods Strain WCHECl-1060 was subjected to WGS to identify known colistin resistance mechanisms. Tn5 insertional mutagenesis, gene knockout and complementation and shotgun cloning were employed to investigate unknown colistin heteroresistance mechanisms. RNA sequencing was performed to link the newly identified mechanism with known ones. Results We showed that the phoP gene [encoding part of the PhoP-PhoQ two-component system (TCS)], the dedA(Ecl) gene (encoding an inner membrane protein of the DedA family) and the tolC gene (encoding part of the AcrAB-TolC efflux pump) are required for colistin heteroresistance. We identified a new gene, ecr, encoding a 72 amino acid transmembrane protein, which was able to mediate colistin heteroresistance. We then performed RNA sequencing and transcriptome analysis and found that in the presence of ecr the expression of phoP and the arnBCADTEF operon, which synthesizes and transfers l-Ara4N to lipid A, was increased significantly. Conclusions The small protein encoded by ecr represents a new colistin heteroresistance mechanism and is likely to mediate colistin heteroresistance via the PhoP-PhoQ TCS to act on the arnBCADTEF operon.