Objectives/Hypothesis: Tenascin-C (Tnc) is an extracellular matrix (ECM) glycoprotein that plays a vital role in wound healing and fibrotic disease. Tnc is highly upregulated soon after vocal fold injury, but its function in the vocal fold has not yet been defined. In this study, we investigated Tnc expression in a rat vocal fold injury model in vivo and its roles in fibroblasts in vitro. Study Design: In vivo and in vitro. Methods: Tnc mRNA and protein expression levels were quantified on days 3, 7, 14, 28, and 56 after vocal fold injury in Sprague-Dawley rats. In vitro, immunocytochemistry, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR) analyses were performed in primary rat vocal fold fibroblasts following Tnc or transforming growth factor (TGF)-beta 1 stimulation to investigate the phenotypic effects. Results: Tnc mRNA and protein expression was upregulated dramatically on days 3 and 7 after injury, and significant differences were observed by qRT-PCR (P < .01). In vitro, Tnc significantly stimulated the migration of primary vocal fold fibroblasts. Following incubation with Tnc for 72 hours, alpha-smooth muscle actin, collagen I, and fibronectin expression was significantly upregulated (P < .05). TGF-beta 1 increased Tnc expression in a time-dependent manner, and a mothers against decapentaplegic homolog 3 (SMAD3) inhibitor attenuated this TGF-beta 1-induced stimulation. Conclusions: Tnc was highly upregulated during the early postinjury period in vivo and promoted vocal fold fibroblast migration, transdifferentiation, and ECM protein synthesis in vitro. Tnc was induced by TGF-beta 1 in a SMAD3-dependent manner. Transient expression of Tnc is likely to promote regeneration, but its potential role in fibrosis requires further study.