Polymyxins are polycationic antimicrobial peptides used as last-resort antibiotics for treating multidrug-resistant Gram-negative bacterial infections. However, resistance to polymyxins has increased, with some bacteria, like Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii, developing acquired resistance, while others, such as Proteus spp., Serratia spp., and Burkholderia spp., are naturally resistant. This review summarizes the mechanisms bacteria use to resist polymyxins, including lipopolysaccharide (LPS) modifications, efflux pumps, capsule formation, and outer membrane protein overexpression. LPS modifications, such as adding phosphoethanolamine (PEtN) and 4-amino-4-deoxy-L-arabinose (L-Ara4N) to lipid A, reduce the negative charge of LPS, decreasing its binding to polymyxins. The PmrA/PmrB and PhoP/PhoQ two-component systems regulate these modifications, with mutations in these systems leading to constitutive activation and increased resistance. In Salmonella, mutations in pmrA and pmrB genes lead to increased colistin resistance, while in Klebsiella pneumoniae, mutations in mgrB also contribute to resistance. In Acinetobacter baumannii, mutations in pmrA and pmrB, as well as the loss of lipid A biosynthesis genes, lead to resistance. The capsule and efflux pumps also play roles in resistance. The loss of LPSs in some bacteria, such as A. baumannii, results in complete resistance. Colistin resistance in A. baumannii can also be due to heteroresistance, where some bacteria are less resistant. The fitness cost of colistin resistance includes reduced growth, virulence, and clinical invasiveness. Understanding these mechanisms is crucial for developing new treatments for Gram-negative bacterial infections.Polymyxins are polycationic antimicrobial peptides used as last-resort antibiotics for treating multidrug-resistant Gram-negative bacterial infections. However, resistance to polymyxins has increased, with some bacteria, like Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii, developing acquired resistance, while others, such as Proteus spp., Serratia spp., and Burkholderia spp., are naturally resistant. This review summarizes the mechanisms bacteria use to resist polymyxins, including lipopolysaccharide (LPS) modifications, efflux pumps, capsule formation, and outer membrane protein overexpression. LPS modifications, such as adding phosphoethanolamine (PEtN) and 4-amino-4-deoxy-L-arabinose (L-Ara4N) to lipid A, reduce the negative charge of LPS, decreasing its binding to polymyxins. The PmrA/PmrB and PhoP/PhoQ two-component systems regulate these modifications, with mutations in these systems leading to constitutive activation and increased resistance. In Salmonella, mutations in pmrA and pmrB genes lead to increased colistin resistance, while in Klebsiella pneumoniae, mutations in mgrB also contribute to resistance. In Acinetobacter baumannii, mutations in pmrA and pmrB, as well as the loss of lipid A biosynthesis genes, lead to resistance. The capsule and efflux pumps also play roles in resistance. The loss of LPSs in some bacteria, such as A. baumannii, results in complete resistance. Colistin resistance in A. baumannii can also be due to heteroresistance, where some bacteria are less resistant. The fitness cost of colistin resistance includes reduced growth, virulence, and clinical invasiveness. Understanding these mechanisms is crucial for developing new treatments for Gram-negative bacterial infections.