Significance Gram-negative bacteria possess an outer membrane (OM) which reduces antibiotic effectiveness. The OM has a dense outer layer of lipopolysaccharide (LPS), a glycolipid which is directly targeted by the last-resort antibiotic polymyxin B (PmB). However, we lack accurate molecular data on the PmB–OM interaction to inform future drug development. Here we study the PmB–OM interaction in vitro and show that in vivo results are reproduced only when the LPS is in a liquid crystalline phase, which occurs at body temperature. These findings not only explain the temperature dependence of PmB function but also support the notion that bacteria actively control the viscosity of their outer membranes as growth temperatures vary.