Multidrug-resistant Gram-negative bacteria present an urgent and formidable threat to the global public health. Polymyxins have emerged as a last-resort therapy against these 'superbugs'; however, their efficacy against pulmonary infection is poor. In this study, we integrated chemical biology and molecular dynamics simulations to examine how the alveolar lung surfactant significantly reduces polymyxin antibacterial activity. We discovered that lung surfactant is a phospholipid-based permeability barrier against polymyxins, compromising their efficacy against target bacteria. Next, we unraveled the structure-interaction relationship between polymyxins and lung surfactant, elucidating the thermodynamics that govern the penetration of polymyxins through this critical surfactant layer. Moreover, we developed a novel analog, FADDI-235, which exhibited potent activity against Gram-negative bacteria, both in the presence and absence of lung surfactant. These findings shed new light on the sequestration mechanism of lung surfactant on polymyxins and importantly pave the way for the rational design of new-generation lipopeptide antibiotics to effectively treat Gram-negative bacterial pneumonia.