Context: TGF-β plays a central role in hypertrophic scar (HS) formation and development.Objective: This study investigated the role of a TGF-β antagonist peptide in inhibiting fibrotic behavior of human HS-derived fibroblasts (HSFs).Materials and methods: HSFs were seeded at a density of 3.1 × 104/cm2 and were subjected to treatment of peptide antagonist (30 μM) or TGF-β receptor inhibitor LY2109761 (10 μM) or without treatment followed by the analyses of quantitative PCR, Elisa, in vitro wounding and fibroblast-populated collagen lattice (FPCL) assays.Results: qPCR and Elisa analyses showed that the peptide could, respectively, reduce the gene (at 48 h) and protein (at 72 h) expression levels of collagen I (86 ± 4.8%; 56.6 ± 7.3%), collagen III (73 ± 10.7%; 43.7 ± 7.2%), fibronectin (90 ± 8.9%; 21.1 ± 2.8%), and TGF-β1 (85 ± 9.3%; 25.0 ± 9.4%) as opposed to the non-treated group (p < 0.05), as the LY2109761 group similarly did. Cell proliferation was also significantly inhibited at day 5 (CCK-8 assay) by both peptide and LY2109761 treatments compared with the non-treated group (p < 0.05). The peptide also significantly inhibited cell migration as opposed to blank control at 24 h (43 ± 6.7% versus 60 ± 2.1%, p < 0.05) and at 48 h (63.9 ± 3.1% versus 95 ± 4.1%, p < 0.05). Similar to LY2109761, the peptide antagonist significantly reduced HS FPCL contraction compared with the non-treated group with significant differences in surface area at 48 h (0.71 ± 0.06 cm2 versus 0.51 ± 0.06 cm2, p < 0.05) and at 72 h (0.65 ± 0.02 cm2 versus 0.42 ± 0.01 cm2, p < 0.05).Conclusion: The TGF-β antagonist peptide may serve as an important drug for HS prevention and reduction given the obvious benefits of good biosafety, low cost, and easy manufacture and delivery.