Micropatterning techniques were widely applied to manipulate morphology of colony and to reveal the critical role of spatial factors in tumorigenesis and gastrulation. However, the effect of morphological cues on bioactive substances treatment during these biological processes was still not clear. Therefore, the influence of colony's morphology on cellular uptake capacity of insoluble bioactive substance should be another essential factor in biomolecular induced cellular behaviors' variation. In this study, PDMS stencils were applied to control colonies' size, and relationship between cellular uptake capacity of nanoparticles and morphological cues of colonies was analyzed. Consequently, benefited from more nanoparticles within micropatterns, the larger colonies endocytosed more nanoparticles. Additionally, concentrated cells with high cell seeding density or located at the peripheral region of micropatterned colonies have higher cellular uptake capacity. The improved cellular uptake capacity at the peripheral region was mainly caused by the uneven distribution of nanoparticles. In addition, the high cell density promoted cellular uptake was benefited from the cortical actin accelerated clathrin-mediated endocytosis in circular-shaped cells.