The adsorption patterns of human serum albumin (HSA) on the monolayers of dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol mixtures were studied by using the tapping mode atomic force microscopy (AFM). The pure DPPC and DPPC/cholesterol monolayers with different packing density were deposited on alkylated substrates by using the Langmuir-Blodgett technique, with the varying concentrations of cholesterol. The structures of solid supported pure DPPC and solid supported DPPC/cholesterol monolayers were analyzed by using AFM, ellipsometry, and cyclic voltammetry. The results indicated that the 10-30 mol % cholesterol mixed monolayers had densely packed and ordered structures, but the 50 mol % cholesterol mixed monolayer had irregular and disordered structures. When the DPPC and DPPC/cholesterol monolayers were exposed to a 0.1 mg/mL solution of HSA, the adsorption patterns of albumin indicated that the pure DPPC and the 50 mol % cholesterol mixed monolayers greatly activated protein adsorption, whereas the 10-30 mol % cholesterol mixed monolayers strongly suppressed protein adsorption due to highly packed phosphocholine groups. Highly packed DPPC molecules in the monolayer were vertically oriented and the protein-resistant neutral phosphocholine groups were in direct contact with proteins, thereby reducing protein adsorption. Therefore, the protein adsorption patterns greatly depended on the condensing effect of cholesterol in the DPPC monolayer.