A large number of extracellular matrix proteins have been found in phosphorylated states, yet little is known about how the phosphorylation of extracellular matrix proteins might affect cell functions. We thus tested the hypothesis whether the phosphorylation of fibronectin, a major adhesion protein, affects cell behavior. Controlled in vitro phosphorylation of fibronectin by a casein kinase II (CKII) significantly upregulated cell traction forces and total strain energy generated by fibroblasts on nanopillar arrays, and consequently other elementary cell functions including cell spreading and metabolic activity. Mass spectrometry of plasma fibronectin from healthy human donors then identified a constitutively phosphorylated site in the C-terminus, and numerous other residues that became phosphorylated by the CKII kinase in vitro. Our findings open up novel strategies for translational applications including targeting diseased ECM, or to develop assays that probe the phosphorylation state of the ECM or blood as potential cancer markers.