Although molecular and physical mechanisms of fibroblast matrix assembly have been widely investigated, the role of adhesive ligand presentation on matrix assembly has only been recently probed (Pereira et al. Tissue Eng., 2007). In the present study, various-sized albumin-derived nanocarriers (ANCs) were fabricated as nanoscale organization units for functionalization with the cell adhesion domain of fibronectin. The adhesion, morphology, and matrix assembly of human dermal fibroblasts were compared on substrate-deposited, ligand-ANCs of varying size. At early time points, fibroblast attachment, stress fiber formation, and spreading were higher on functionalized, larger-sized carriers than on smaller carriers. Matrix assembly was greatest at the highest ligand density on larger nanocarriers but was undetectable at the same ligand density on smaller carriers. Tracking of fluorophore-encapsulated ANCs showed that larger carriers were displaced less than smaller carriers and that atomic force microscopy of ligand-ANCs binding to adherent cells demonstrated that the larger ligand-ANCs required larger dissociation forces. Taken together, these data suggest that the greater inertia of larger adhesive nanocarriers may generate more cellular tension, which in turn, promotes up-regulation of matrix assembly. Thus, the size of the nanocarrier and the density of ligand on that nanocarrier combine to dictate the early kinetics of fibroblast matrix assembly. These insights may be useful for understanding cell-matrix interactions, as well as for development of bioactive materials with defined cell-adhesive activities such as wound repair and matrix remodeling events.