Adherent cells respond to changes in their microenvironment by generating internal forces that are transferred to the substrate to which they are attached. Manipulation of cellular signaling pathways also influences the response to these changes and can direct cells to pathological outcomes. C-type natriuretic peptide (CNP), through the NPR-B receptor, has been shown to protect quiescent fibroblasts in heart valves from differentiating into myofibroblasts that contribute to development of calcific aortic valve disease (CAVD). Here, I implemented and validated a method for quantifying cellular traction forces in vitro, and used this method to characterize fibroblasts’ response to haploinsufficiency of Npr2, the gene that encodes NPR-B. I showed that CNP significantly lowered traction forces in Npr^2+/+ fibroblasts, but not Npr2^+/- fibroblasts. These studies demonstrate a novel effect of CNP on fibroblast biomechanical function, adding to our knowledge of the mechanisms by which CNP may protect against CAVD.