Mediastinal Müllerian cysts are rare and are composed of ciliated Müllerian-type tubal epithelium. Preoperative diagnosis of this cyst is usually difficult owing to the local anatomy. We report a case of a Müllerian cyst in the mediastinum of a 25-year-old female patient that was resected by robotassisted thoracoscopic surgery (RATS) without rupturing the cyst. The tumor was a monolocular cyst covered with a thin capsule, and pathological examination showed a thin-walled cyst lined by ciliated or nonciliated columnar cells resembling tubal epithelium. Immunohistochemical (IHC) analysis showed positive expression for paired box protein 8 (PAX8), Wilms' tumor 1 (WT-1), and estrogen receptors (ERs) and progesterone receptors (PRs). From the results of IHC analysis of control cases of mediastinal cystic tumors, four types of IHC analysis are quite helpful in obtaining a definitive diagnosis of Müllerian cyst. Although this cyst formation has been considered to be related to hormone imbalance, we suggest that hormone imbalance may be irrelevant to the etiology of Müllerian cyst, considering our case and two previous young cases. According to the fact that there is no significant difference between Müllerian cyst and non-Müllerian cyst in hematoxylin and eosin (HE) staining, a Mülleriancyst should always be considered in patients with a posterior mediastinal cyst, especially when it is adjacent to a thoracic vertebral body.