Antibiotics play a pivotal role in modern medicine for the treatment of bacterial infection in patients. Membrane defines the boundary between single cell and its environment and is a main target for antibacterial agents. To better understand the mechanism of antibiotics action on microbes, we utilized liposome as membrane mimic model to study antibiotics interaction with bacterial membrane by variety of biophysical methods. Isothermal calorimetry and fluorescence photometry experiments were performed to examine interaction between antibiotics and liposome. We found that vancomycin, one of the most important antibiotics for the treatment of serious infections by gram-positive bacteria, binds to the liposome. The association between the drug and the liposome does not involve the tail part of the lipids. Moreover, the binding affinity increases along with the increment of liposome size. Of three major lipid components, phosphatidylglycerol is the preferential target for vancomycin binding. We also showed that vancomycin associates with vesicle derived from Staphylococcus aureus membrane in a similar manner as the binding to liposome. Our data suggested that vancomycin associates with bacterial membrane through direct interaction with lipid head groups with the extent of the association depending very much on specific type of lipids and curvature of local membrane structure.