Research data underpinning publication Polymyxin B lethality requires energy-dependent outer membrane disruption, accepted for publication at Nature Microbiology (2025), and manuscript deposited at https://www.biorxiv.org/content/10.1101/2025.04.16.649083v1.Polymyxin antibiotics target lipopolysaccharide (LPS) in both membranes of the bacterial cell envelope, leading to bacterial killing through a mechanism that remains poorly understood. Here, we demonstrate that metabolic activity is essential for polymyxin lethality and leverage this insight to determine its mode of action. Polymyxin B (PmB) efficiently killed exponential phase E. coli but was unable to eliminate stationary phase cells unless a carbon source was available. Antibiotic lethality correlated with surface protrusions, LPS loss from the outer membrane (OM), and a corresponding reduction in barrier function, processes that required LPS synthesis and transport, but were blocked by the MCR-1 polymyxin resistance determinant. While the energy-dependent OM disruption was not directly lethal, it facilitated PmB access to the inner membrane (IM), which the antibiotic permeabilised in an energy-independent manner, leading to cell death. This work reveals how metabolic inactivity confers tolerance of a clinically important, membrane-targeting antibiotic, leading to new insight into mechanism of action.Data include microscopy data (in generic image format or if not, accessible by open-source software, https://gwyddion.net/) and spreadsheets with tabulated data for plots shown in figures (quantification of cell death, fluorescence, etc.).