Objective: Situs inversus totalis (SIT) is a relatively rare congenital abnormality in which the major thoracic and abdominal visceral organs are reversed from their usual positions. Here, we present a first case of SIT patient with lung cancer, in which da Vinci robot-assisted thoracic surgery (RATS) was performed successfully. Method: The Mimics Medical 21.0 was used to construct three-dimensional (3D) reconstruction. Three-port RATS was conducted to treat this SIT patient with lung cancer. Result: A 66-year old patient, previously diagnosed with SIT since childhood, came to our hospital with two pulmonary nodules in left upper lobe. The bigger one had increased somewhat for the last 2 years of follow-up. Software Mimics was preoperatively carried out to analyze anatomical variations. RATS was conducted to complete left upper lobectomy and left middle wedge resection. The patient had no intraoperative complications and was discharged day 5 postoperatively. Conclusion: This is the first report of a successful robot-assisted lung cancer resection in a patient with SIT. In such challenging cases as lung cancer and rare anomaly as SIT, RATS is advantageous and suitable with the help of 3D reconstruction, which can identify the anatomical abnormalities and facilitate the surgical procedures.