The emergence of multidrug resistance in bacteria has reached alarming levels. To solve this growing problem, discovery of novel cellular targets or pathways important for antimicrobial resistance is urgently needed. In this study, we explored how the alternative sigma factor, RpoE, protects
Escherichia coliO157 against the toxic effects of the polycationic antimicrobial agent, chlorhexidine (CHX). Susceptibility of this organism to CHX was found to directly correlate to the growth rate, with the faster replicating wild‐type being more susceptible to CHX than its more slowly replicating Δ rpoEO157 mutant. Once the wild‐type and rpoEmutant strains had undergone growth arrest (entered the stationary growth phase), their resistance to CHX became entirely dependent on the functionality of RpoE. The RpoE regulon plays a critical role in maintaining the integrity of the asymmetric lipid bilayer of E. coli, thereby preventing the intracellular accumulation of CHX. Finally, using a single‐cell, high‐resolution, synchrotron‐based approach, we discovered a subpopulation of the rpoEmutant strain with no detectable intracellular CHX, a predominant characteristic of the wild‐type CHX‐resistant population. This finding reveals a role of phenotypic heterogeneity in antimicrobial resistance.