The two-dimensional (2D) assembly of the protein streptavidin on a biotin-bearing lipid monolayer was studied as a function of the surface density of biotin, a protein-binding ligand, by means of in situ X-ray scattering and optical Brewster angle microscopy measurements at the liquid–vapor interface. Although this model system has been studied extensively, the relationship between the surface biotin density and the adsorption, 2D phase behavior, and binding state of streptavidin has yet to be determined quantitatively. The observed equilibrium phase behavior provides direct structural evidence that the 2D crystallization of the lipid-bound streptavidin occurs as a density-driven first-order phase transition. The minimum biotin density required for the 2D crystallization of streptavidin is found to be remarkably close to the density of the ligand-binding sites in the protein crystal. Moreover, both above and below this transition, the observed biotin-density dependence of protein adsorption is well described by the binding of biotin-bearing lipids at both of the two available sites per streptavidin molecule. These results imply that even in the low-density noncrystalline phase, the bound proteins share a common, fixed orientation relative to the surface normal, and that the 2D crystallization occurs when the lateral protein density reaches 50–70% of the 2D crystal density. This study demonstrates that in addition to a well-defined molecular orientation, high lateral packing density is essential to the 2D crystallization of proteins.