Radiation therapy (RT) is a typical treatment for head and neck cancers. However, prolonged irradiation of the esophagus can cause esophageal fibrosis due to increased reactive oxygen species and pro-inflammatory cytokines. The objective of this study was to determine whether myogenic gene transfected-MSCs could ameliorate damage to esophageal muscles in a mouse model of radiation-induced esophageal fibrosis. We cloned esophageal myogenic genes (MyoD, MyoG, and Myf6) using plasmid DNA. Afterward, myogenic genes were transfected into hMSCs using electroporation. Gene transfer efficiency, stemness, and myogenic gene profile were examined using FACS, qPCR, and RNA sequencing. In vivo efficacy of gene-transfected hMSCs was demonstrated through histological and gene expression analyses using a radiation-induced esophageal fibrosis animal model. We have confirmed that the gene transfer efficiency was high (approximately 75%). Pluripotency levels in gene transfected-MSCs were significantly decreased compared to those in the control (vector). Particularly, myogenesis-related genes such as OAS2, OAS3, and HSPA1A were overexpressed in the group transfected with three genes. At 4 weeks after injection, it was found that thickness collagen layer and esophageal muscle in MSCs transfected with all three genes were significantly reduced compared to those in the saline group. Muscularis mucosa was observed prominently in the gene combination group. Moreover, expression levels of myogenin, Myf6, calponin, and SM22α known to be specific markers of esophageal muscles tended to increase in the group transfected with three genes. Therefore, using gene transfected MSCs has potential as a promising therapy against radiation-induced esophageal fibrosis.