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A Müllerian duct cyst is a rare epithelial-lined cyst originating from remnants of the Müllerian (paramesonephric) duct. While these cysts are typically found along the lateral or posterior vaginal wall in females or near the prostate in males, posterior mediastinal occurrence is exceedingly rare. Accurate preoperative diagnosis is challenging, as imaging findings often resemble those of more common posterior mediastinal lesions such as neurogenic tumors or bronchogenic cysts. Surgical resection is usually required for definitive diagnosis and treatment. Video-assisted thoracic surgery (VATS) via the confronting upside-down monitor setting is a novel approach that offers improved visualization and ergonomics, but has not previously been reported for resection of a posterior mediastinal Müllerian duct cyst. A 44-year-old woman was referred following the incidental detection of a right posterior mediastinal mass on chest radiography. She was asymptomatic, with normal laboratory results including tumor markers. Contrast-enhanced computed tomography (CECT) revealed a well-defined, non-enhancing mass adjacent to the right T5-T6 vertebral bodies, measuring 3.3 × 1.8 × 2.8 cm. Magnetic resonance imaging (MRI) showed a cystic lesion with high T2 signal intensity. A neurogenic tumor or bronchogenic cyst was suspected, and surgical resection was performed using a four-port VATS approach with the confronting upside-down monitor setting. Intraoperatively, a smooth-surfaced cyst originating from the T5 vertebral body was identified and resected en bloc with the adherent fifth intercostal vein. Histopathology revealed a cyst lined by cuboidal epithelium, positive for Claudin4, estrogen receptor, Wilms' Tumor 1 (WT1), and Paired box gene 8 (PAX8), and negative for Calretinin, findings consistent with a Müllerian duct cyst. The patient's postoperative course was uneventful, and she was discharged on postoperative day two. Posterior mediastinal Müllerian duct cysts, though rare, should be considered in the differential diagnosis of mediastinal cysts in perimenopausal women. Definitive diagnosis requires histopathological and immunohistochemical evaluation. VATS via the confronting upside-down monitor setting provides direct visualization, minimal instrument interference, and improved spatial orientation, making it a valuable surgical option, particularly for lesions in close proximity to the intercostal vessels or the vertebral column. This case represents the first reported resection of a posterior mediastinal Müllerian duct cyst using this approach. .