Purpose The interactions of poly(ethylene oxide)-co-poly(propylene oxide) tri-block copolymers (PEO-PPO-PEO block copolymers, Pluronics®, Synperonics®, Poloxamers) of differing chemical composition with cell membranes were systematically investigated in order to clarify the mechanisms behind their previously reported various cellular responses. Methods Relationships between the structural components of a defined series of PEO-PPO-PEO block copolymers and i) their interactions with biological membranes; ii) their cytotoxic potential were probed using a combination of haemolysis studies and cytotoxicity assays in the Caco-2 and HMEC-1 cell lines. Results The length of the PPO block as well as the PEO/PPO ratio were determinants of their membrane binding constant and cytotoxicity endpoints measured in the MTS and LDH assays. Similar 2D parabolic relationships were found between polymer composition and their affinity for membranes or their cytotoxicity potential. Cytotoxicity was related to the ability of the copolymers to form ion transversable pores within the cell membrane. Conclusions The data suggest a link between the affinity of certain Pluronics for biological membranes and their cellular adverse effects. This first cell-based investigation of the interactions of Pluronics with biological membranes is an important step towards unravelling the complex mechanisms which govern the biological effects of widely used amphiphilic materials.