The objective of this case study is to present our experience of a surgical approach for vaginal agenesis using an acellular porcine small intestinal submucosa (SIS) graft. The present report involved 2 patients diagnosed as having vaginal agenesis due to Meyer-von-Rokitansky-Küster-Hauser syndrome. The operation procedure involved the creation of a neovaginal tunnel and then a mold wrapped with the SIS graft was placed in the neovagina. The duration of surgery was less than 45 min with minimal blood loss and no operative and postoperative complications. Epithelialization of the neovagina was achieved within 2 months after surgery. The neovagina created with this procedure was the same as that of a normal adult vagina histologically and physiologically. In conclusion, the creation of a neovagina using a SIS graft resulted in a favorable outcome and this approach may be a potential alternative to the management of vaginal agenesis.