Background: Hypertrophic scar (HS) is a fibroproliferative disease characterized by the overproduction of extracellular matrix, cell over-proliferation, enhanced angiogenesis and differentiation of fibroblasts. Although there has been extensive research on botulinum toxin type A (BTX) treatment for the prevention of HS formation, its effectiveness for the attenuation of skin fibrosis and the related mechanism are unclear. Objectives: BTX treatment on HS would show suppressive effect on scar-related factors. Methods: Human scar fibroblasts (HSFs) were cultured and stimulated with BTX. MTS, scratch, quantitative polymerase chain reaction, enzyme-linked immunosorbent, and western blot assays were performed to detect changes in fibroblast proliferation, migration, and gene/protein expression of pro-fibrotic factors. Results: The proliferation and migration of BTX-treated HSFs were decreased compared to those in untreated controls. The mRNA and protein expression of pro-fibrotic factors was inhibited, whereas JNK phosphorylation was activated. Blocking the JNK pathway rescued the inhibitory effects on HSF proliferation and the production of pro-fibrotic factors. Conclusion: This study showed that BTX has a suppressive effect on ECM production and scar-related factors in HSFs in vitro. Moreover, regulation of JNK signaling played an important role in this process. Our results provide some theoretical basis for HS treatment.