The interaction between a monosaccharide (fructose, mannose, glucose, or galactose) and a phospholipid (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)) molecular layer was investigated experimentally to understand the non-genomic effects on biomembranes. The surface pressure (π)–surface area (A) isotherm of the phospholipid monolayer at an air–water interface was changed characteristically depending on the kind of monosaccharide used. Attenuated total reflection Fourier transform spectroscopy and nuclear magnetic resonance spectroscopy were used to evaluate the interaction between DMPC and monosaccharide molecules.