Culturing cells on gradient nanopatterns provides a useful tool to explore cellular adhesion to mimics of the extracellular matrix or screen for cellular responses to nanopatterns. A method is presented to fabricate complex gradient protein patterns based on hole-mask colloidal lithography, which can generate nanopatterns in multiple materials and of multiple shapes. Gradients of gold structures were functionalized to form gradients of protein nanopatterns of different shapes (bars, dot pairs, and rings), where a key parameter was systematically varied in each gradient. Cells were grown on vitronectin nanopatterns, showing differential adhesion (spread area/focal adhesion size) along the gradients.