As a kind of membrane-active lipopeptide antibiotics, polymyxins are the last-line therapy against multidrug-resistant Gram-negative pathogens. Through interacting with the lipopolysaccharide molecules, polymyxins disorganize the structure of bacterial outer membrane, and finally lead to the cell death. Nevertheless, the precise mechanisms of polymyxin pharmacology remain largely unknown, which is mainly due to the limited ability of current biochemical and structural approaches to characterize the interaction between cell membranes and drugs. This in turn significantly hinders the design and development of new-generation polymyxins. In recent years, molecular dynamics simulations have been successfully applied in the field of polymyxin pharmacology. In particular, a series of simulation models, including bacterial membranes-polymyxins, and human cell membrane-polymyxins, has been developed and tested. Previous studies have shown that polymyxin adopted a unique folded conformation in bacterial outer membrane, which played a key role in the antimicrobial activity of polymyxins. Further, various lipopolysaccharide modifications could change the structural and physical properties of bacterial outer membrane and thereby confer polymyxin resistance to bacteria. Moreover, recent studies revealed that polymyxins may disrupt the membrane of renal tubular cells, and also attenuate the function of different ion channels, which provide a clue to understand the detailed mechanism of polymyxin-induced nephrotoxicity. In this review, we summarized the applications of molecular dynamics simulations in the interaction of polymyxins with different biological membranes, with the aim to refresh our understanding of the link between polymyxin pharmacology and cell membranes and to provide mechanistic guides for the future design of novel antimicrobial drugs.