BACKGROUND The ideal scaffold material should provide immediate capacity to bear mechanical loads and also permit eventual resorption and replacement with native tissue of similar mechanical integrity. Scaffold characteristics such as fiber diameter provide environmental cues that can influence cell function and differentiation. In this study, the impact of fiber diameter of scaffolds constructed from a tyrosine-based bioresorbable polymer on cellular response was investigated. METHODS Electrospun bioresorbable poly(desamino tyrosyl-tyrosine ethyl ester carbonate) scaffolds composed of microfibers or nanofibers were constructed and seeded with human dermal fibroblasts. The impact of fiber diameter on actin cytoskeletal morphology, focal adhesion size, fibronectin matrix assembly, and cell proliferation was evaluated using immunofluorescent microscopy and computer-assisted image analysis. RESULTS Actin stress fibers were more easily observed in cells on microfiber scaffolds compared with those on nanofiber scaffolds. Cells on nanofiber scaffolds developed smaller focal adhesion complexes compared with those on microfiber scaffolds (p < 0.0001). The temporal patterns of fibronectin matrix assembly were affected by scaffold fiber diameter, with cells on microfiber scaffolds showing a delayed response in dense fibril formation compared with nanofiber scaffolds. Cells on nanofiber scaffolds showed higher proliferation compared with microfiber scaffolds at time points under 1 week (p < 0.01), but by 2 weeks significantly higher cell proliferation was observed on microfiber scaffolds (p < 0.01). CONCLUSIONS The fiber diameter of bioresorbable scaffolds can significantly influence cell response and suggests that the ability of scaffolds to elicit consistent biological responses depends on factors beyond scaffold composition. Such findings have important implications for the design of clinically useful engineered constructs.