Tissue engineering has revealed the potential to regenerate injured vocal folds, and identification of the most suitable seed cells has remained a hot topic of research. The aim of this study was to implant fibroblast-like cells differentiated from adipose-derived mesenchymal stem cells (ADSCs) in a canine acute vocal fold wound model. We then sought to characterize changes in the extracellular matrix (ECM) proteins of vocal fold lamina propria. For this purpose, ADSCs were induced to differentiate into fibroblasts under the regulation of connective tissue growth factor in vitro. Cell surface proteins were identified by immunofluorescence staining. Thirty vocal folds of 17 canines were injured by localized resection and injected with fibroblast-like cells (differentiated ADSCs, dADSCs), ADSCs or vocal fold fibroblasts (VFFs). The morphology of vocal folds was observed, and the characteristics of ECM protein components (collagen, elastin, hyaluronic acid, decorin and fibronectin) were evaluated by immunofluorescence staining from 15 days to 6 months following implantation. The results showed that in vitro, the dADSCs showed morphology and cell surface protein expression similar to those of VFFs. After implantation in vivo, the surfaces of the recipient vocal folds became almost smooth in the dADSCs and ADSCs groups at 6 months but remained slightly concave and stiff in the VFFs group. The elastin fluorescence intensity increased significantly and was maintained at a high level in the dADSCs group. The collagen fluorescence intensity increased slightly in the dADSCs and ADSCs groups, whereas it demonstrated a more irregular arrangement in the VFFs group. The fluorescence intensity of hyaluronic acid, decorin and fibronectin was similar between the three implanted groups. These results indicated that dADSCs may confer an advantage for vocal fold wound healing. Furthermore, dADSCs have the ability to secrete ECM components in vivo, particularly elastin, which may be beneficial for vocal fold vibration recovery.