Diffusivity of Cholera toxin subunit B (CTX) bind to GM1 molecules in 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC) bilayer was observed by total internal reflection microscope (TIRFM). Numerical analysis based on mean square displacement revealed the presence of higher (D = 0.4 μm²/s) and lower (D < 0.1 μm²/s) diffusive fractions, both of which were an ordered of slower compared to dye-labeled lipid (D = 3.5 μm²/s). The observed difference in CTX-GM1 diffusivity reflects the characteristics of the multivalent CTX-GM1 binding properties. [DOI: 10.1380/ejssnt.2009.74]