Hypertrophic scar (HS) is a common type of dermatosis. Botulinum toxin type A (BTXA) can exert an anti-HS effect; however, the regulatory mechanisms underlying this effect remain unclear. Thus, the aim of this study was to examine the effects of BTXA on phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression and the fibroblast phenotypic transformation induced by transforming growth factor (TGF)-β1, which is an important regulatory factor involved in the process of HS. For this purpose, fibroblasts were treated with various concentrations of BTXA and then treated with 10 ng/ml of TGF-β1 with gradient concentrations of BTXA. The proliferation and apoptosis of fibroblasts were measured by cell counting kit-8 assay (CCK-8) and flow cytometry, respectively. PTEN methylation was analyzed by methylation-specific PCR (MSP) and DNA methyltransferase (DNMT) activity was determined using a corresponding kit. RT-qPCR and western blot analysis were performed to detect the transcription and translation levels. The results revealed that BTXA suppressed the proliferation and increased the apoptosis of fibroblasts treated with TGF-β1 in a dose-dependent manner. BTXA in combination with TGF-β1 suppressed the expression of molecules related to the extracellular matrix (ECM), epithelial-mesenchymal transition (EMT) and apoptosis. BTXA reduced the PTEN methylation level and downregulated the expression levels of methylation-associated genes. BTXA also inhibited the phosphorylation of phosphoinositide 3-kinase (PI3K) and Akt. On the whole, the findings of this study indicate that BTXA may inhibit fibroblast phenotypic transformation by regulating PTEN methylation and the phosphorylation of related pathways. The findings of this study can provide a theoretical basis for HS treatment.