Extracellular matrix (ECM) proteins, especially fibronectin, are critical pathological characteristics of renal fibrosis and contribute to the progression of chronic renal disease. Recent studies showed that mechanical stretch is associated with the development of renal fibrosis in a murine model of unilateral ureteral obstruction (UUO). However, the molecular events responsible for stretch‐induced renal fibrosis, following UUO, have yet to be determined. In this study, we investigated the mechanism by which mechanical stretch, an in vitro model for chronic obstructive nephropathy, regulates fibronectin expression in cultured human proximal tubular epithelium (HK‐2) cells. Mechanical stretch up‐regulated fibronectin expression and activated STAT3 in a time‐dependent manner. Stretch‐induced fibronectin were suppressed by a STAT3 inhibitor, S3I‐201 and by small interfering RNA (siRNA) targeting human STAT3 (STAT3 siRNA). Similarly, fibronectin expression and STAT3 activation induced by mechanical stretch was suppressed by the Src family kinase inhibitor PP2 and by transfection of HK‐2 cells with a dominant‐negative mutant of c‐Src (DN‐Src), whereas PP3, an inactive analogue of PP2, had no significant effect. This work demonstrates, for the first time, a role for c‐Src and STAT3 signaling pathways in stretch‐induced renal fibrosis.