Increasing antibiotic resistance in Gram-negative bacteria, particularly in Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae, presents a global medical challenge. No new antibiotics will be available for these superbugs in the near future due to the dry antibiotic discovery pipeline. Colistin and polymyxin B are increasingly used as the last-line therapeutic options for treatment of infections caused by multidrug-resistant Gram-negative bacteria. This article surveys the significant progress over the last decade in understanding polymyxin chemistry, mechanisms of antibacterial activity and resistance, structure-activity relationships and pharmacokinetics/pharmacodynamics. In the Bad Bugs, No Drugs era, we must pursue structure-activity relationship-based approaches to develop novel polymyxin-like lipopeptides targeting polymyxin-resistant Gram-negative superbugs . Before new antibiotics become available, we must optimize the clinical use of polymyxins through the application of pharmacokinetic/pharmacodynamic principles, thereby minimizing the development of resistance.