Pseudomonas aeruginosa is highly versatile gram-negative pathogen and accounts for high morbidity and mortality rates in cystic fibrosis (CF) patients and other immunocompromised individuals. The infamous resistance of this pathogen to multiple currently available antibacterial agents and the weak pipeline for effective antibiotic agents has accelerated the development of multidrug-resistant (MDR) P. aeruginosa strains leading to rejuvenated clinical interest in the “last resort” cationic antimicrobial agent- polymyxins. However, there are increasing reports of resistance to polymyxins in P. aeruginosa. This is a serious therapeutic challenge for treating devastating infections caused by this pathogen. The resistance to polymyxins is demonstrated to be multifactorial and factors conferring resistance to polymyxins at physiologically relevant conditions remains to be poorly understood. Here we show that the presence of physiological levels of calcium (Ca²⁺) in CF lungs enhances resistance in P. aeruginosa to polymyxin B (Pol B) in both sessile and planktonic environments. Several two-component systems earlier identified to control resistance to Pol B in P. aeruginosa including PhoPQ, PmrAB and ParRS, shows no significant involvement in this resistance indicating the involvement of other novel Ca²⁺-dependent mechanisms. In support of this hypothesis, the transcription of two component systems and several other genes involved in Pol B resistance mechanisms described thus far, are downregulated in the presence of increased Ca²⁺ levels. Through random chemical mutagenesis, we identified three genes contributing to Ca²⁺-induced resistance: PA2803, PA3237, and PA5317. Genome-wide RNA-Seq analyses revealed that the transcription of PA2803 and PA3237 is induced by elevated Ca²⁺. Further, both the outer membrane and inner membrane permeability increases during growth at elevated Ca²⁺. Proteomic and transcriptomic profiling uncovered several Ca²⁺-dependent differences in the membrane structure, including Wzz, PhoP/Q, and OprE. Currently, we aim to elucidate the Ca²⁺-dependent membrane lipid modifications that confer resistance. This will improve our understanding of the molecular mechanisms responsible for Ca²⁺-induced PolB resistance in P. aeruginosa.