Abstract: Fluorescent polystyrene nanospheres (PS) were used to explore the impact of substrate stiffness on cell uptake of nanoparticles in the breast cancer cells. Polyacrylamide (PAA) gels with varying stiffness were prepared by photopolymerization, and type I rat tail collagen was covalently conjugated on the surface of PAA gels to facilitate cell adhesion. Type I rat tail collagen was also used to fabricate collagen gels for 3D cell culture. Cells of human breast cancer cell line MCF-7 were incubated in the 2D culture on PAA gels and 3D culture within collagen gels. Next, nanospheres of 20 nm and 50 nm polystyrene were applied to MCF-7 cells in the 2D or 3D cultures. Cell morphology and uptake efficiency were observed with confocal microscopy. Our study demonstrates that substrate stiffness differentially regulated the cell morphology as well as the cell uptake behavior of polystyrene nanospheres in MCF-7 cells under 2D or 3D culture conditions.