We seek to elucidate the role of nanoscale substrate features that can elicit fibroblast-mediated matrix assembly. We fabricated albumin nanoparticles (ANPs) of various sizes (30-125 nm) that were functionalized with fragments of fibronectin (FNf), a ligand containing motifs critical for cell binding. Cells seeded on 125nm FNf-ANPs exhibited the highest levels of attachment and were more elongated in phenotype compared to cells cultured on smaller sized FNf-ANPs or FNf at the same net FNf concentration. Fibroblasts assembled the greatest amount of matrix when cultured on 125nm FNf-ANPs, which was reduced when seeded on smaller sized FNf-ANPs, FNf adsorbed substrates,, or immobilized FNf-ANPs at the same net FNf concentration. We hypothesized that larger sized mobile FNf-ANCs have more inertia, making them harder to sequester, mimicking a rigid substrate that requires more cytoskeletal tension to contract the substrate. The increase in cytoskeletal tension could activate RhoA, leading to stress fiber formation and cell contraction, and integrin translocation, bringing forth matrix assembly by exposing cryptic FN binding sites on bound soluble FN. These results indicate the nanodisplay of the ligand, size of the ANC, and carrier mobility can be used as parameters when developing novel biomaterials to activate cellular responses.