The present work investigates the interaction of hexadecylbetainate chloride (C16BC), a glycine betaine-based ester with palmitoyl-oleoyl-phosphatidylcholine (POPC), sphingomyelin (SM), and cholesterol (CHOL), three biological relevant lipids present in the outer leaflet of the mammalian plasma membrane. The binding affinity and the mixing behavior between the lipids and C16BC are discussed based on experimental (isothermal titration calorimetry (ITC) and Langmuir film balance) and molecular modeling studies. The results show that the interaction between C16BC and each lipid is thermodynamically favorable and does not affect the integrity of the lipid vesicles. The primary adsorption of C16BC into the lipid film is mainly governed by a hydrophobic effect. Once C16BC is inserted in the lipid film, the polar component of the interaction energy between C16BC and the lipid becomes predominant. Presence of CHOL increases the affinity of C16BC for membrane. This result can be explained by the optimal matching between C16BC and CHOL within the film rather by a change of membrane fluidity due to the presence of CHOL. The interaction between C16BC and SM is also favorable and gives rise to highly stable monolayers probably due to hydrogen bonds between their hydrophilic groups. The interaction of C16BC with POPC is less favorable but does not destabilize the mixed monolayer from a thermodynamic point of view. Interestingly, for all the monolayers investigated, the exclusion surface pressures are above the presumed lateral pressure of the plasma membranes suggesting that C16BC would be able to penetrate into mammalian plasma membranes in vivo. These results may serve as a useful basis in understanding the interaction of C16BC with real membranes.