In this article, we describe the design and manipulation of charged nanomatrices and their application as efficient platforms for modulating cell behaviors. Using electrospraying technology and well designed biomaterials, poly(ɛ-caprolactone; PCL) and polyethylenimine, the negatively charged PCL nanomatrix (nPCL nanomatrix) and the positively charged PCL nanomatrix (pPCL nanomatrix) were fabricated. It was demonstrated that cell adhesion, affinity, and shape were sensitively modulated in negatively and positively charged nanomatrices. Our results showed that the pPCL nanomatrix promoted adhesion of NIH 3T3 fibroblast cells as compared to the nPCL nanomatrix. When fluid shear stress was applied, cell affinity on the pPCL nanomatrix increased even more. NIH 3T3 fibroblast cells adopted a relatively spherical shape on the pPCL nanomatrix while adopting an aligned, narrow shape on the nPCL nanomatrix. It was also found that charged nanomatrices influenced the cross-sectional cell shape. The cross-sectional cell shape on the pPCL nanomatrix was extremely flattened, whereas the cross-sectional cell shape was relatively round on the nPCL nanomatrix and some of the adhered cells floated. We also showed that the surfaces of the nPCL and pPCL nanomatrices adsorbed the different serum proteins. These results collectively demonstrated a combination of environmental factors including nanoscale structure, electrostatic forces, and absorption of biomolecules on charged substrates affected cell response in terms of cellular adhesion and shape.