Imatinib (Gleevec) is a novel chemotherapeutic agent against Bcr-Abl protein tyrozine kinase, playing a crucial role in the therapy of chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GIST). Our study aimed at designing a liposomal imatinib formulation and investigating molecular interactions between lipid and imatinib, within the liposomal membrane. Multilamellar (MLV) and small unilamellar (SUV) vesicles were prepared from alpha-L-dipalmitoyl-phosphatidylcholine (DPPC). The effect of imatinib on the DPPC membrane was studied by electron paramagnetic resonance (EPR) spectroscopy and differential scanning calorimetry (DSC), at pH 5.2 and 9.0, where imatinib is in monocationic and neutral form, respectively. Our results indicate that imatinib interacts mainly with the DPPC head groups, leading to a slight increase in the mobility of the polar headgroups in case of MLVs. Contrary to that, imatinib causes a significant decrease in the fluidity of SUVs, which can be the result of a pH-dependent fusion/fission effect. The size distribution and morphology of liposomes were checked by dynamic light scattering and freeze-fracture electron microscopy. Our results direct attention to investigate the interactions of imatinib with artificial/biological membranes.