The interaction between lipid bilayers and poloxamers has recently attracted much attention, and contradicting effects of poloxamers have been revealed on the integrity of lipid membranes; poloxamers are reported to be either effective sealants or permeabilizers of cell membranes depending on the cell type. To elucidate poloxamer-membrane interactions, we study the roles played by the lipid bilayer phase structure, poloxamer concentration, and temperature using isothermal titration calorimetry (ITC). Our results indicate that the lipid bilayer phase structure plays a critical role in its interaction with poloxamers. Poloxamers are found to partition only into fluid-phase, not gel-phase, membranes. Moreover, the partitioning of poloxamers into fluid-phase liposomes increases with temperature, owing to the enhancement in both the membrane fluidity and the hydrophobicity of the poloxamer at elevated temperatures. Our ITC results also point to a saturation concentration of poloxamers, below which poloxamers can partition into a lipid bilayer without disrupting liposomes and above which they instead disintegrate liposomes into micelles. To address the long-standing question as to whether poloxamers migrate through the lipid bilayer after their adsorption onto preformed liposomes, two ITC protocols are used to cross-validate the distribution of poloxamers over the two leaflets in the bilayer. We find that on a short timescale (approximately 200 min) poloxamers partition into the outer leaflet of the fluid-phase lipid bilayer without much interactions with the inner leaflet. However, on a longer timescale (38 h), they are found to migrate through the bilayer and eventually establish an even distribution in both the inner and outer leaflets of the membrane.