With increasing incidence of antibiotic-resistant 'superbugs' globally and an estimated 10 million deaths per year by 2050, our world is dangerously close to reverting to the pre-antibiotic era. Polymyxins are 'last-resort' antibiotics against many Gram-negative 'superbugs'; however, nephrotoxicity is the major dose-limiting factor. Polymyxins are extensively reabsorbed by, and ultimately cause cell death in kidney tubular cells. However, molecular mechanisms underlying this nephrotoxicity remain poorly defined. Here we employed various molecular techniques to examine the mechanistic pathways which lead to polymyxin B (PMB)-induced cell death in vitro and in vivo. HK-2 (human proximal tubular) cells were treated with PMB (12.5-100 µM) for up to 24 h. Biomarkers for genome and chromosome instability were quantified using fluorescence microscopy. PMB-treated HK-2 cells showed significant increase in micronuclei frequency (up to ~18% total cell population when treated with 100 µM for 24 h, n=500, p<0.05), as well as abnormal mitotic events (over 40% in treated, n=30, p<0.05). Time-course studies were performed using a mouse nephrotoxicity model (cumulative 72 mg/kg over 48 h). Kidneys were collected over 48 hours and investigated for histopathological and DNA damage. Notable increases in γ-H2AX foci were observed in both HK-2 (up to ~44% cells with 5+ foci at 24 h, n=300, p<0.05) and kidney samples (up to ~25%, p<0.05). Consistent with these results, in vitro assays show PMB had high binding affinity to DNA. Altogether, our results indicate PMB has unwanted side-effects, causing DNA damage. This novel mechanistic information may lead to new strategies to overcome the nephrotoxicity of these important last-line antibiotics.