: Better management of lung cancer is crucial for improving the prognosis of such lethal malignancy, and surgical treatment is one of the most important strategies for early lung cancer. Anatomic segmentectomy, in particular, provides the complete resection of lesions with the retention of maximal normal lung tissue. Several different techniques for lung segmentectomy have been described previously, whereas how to perform with concerns of minimal invasiveness and better accessibility for surgeons familiar with single-direction video-assisted thoracic surgery (VATS) remains to be elucidated. In the presented case, we described the applications of our two-micro-portal video-assisted thoracic segmentectomy as well as the plug-in multi-edge cutting method to a patient with a mixed ground-glass nodule in the anterior basal segment of the right lower lobe, and systemic lymphadenectomy was performed following the pathological diagnosis of invasive adenocarcinoma. The patient was discharged after a 4-day well recovery and was without recurrence or metastasis after 6 months of follow-up. Considering that the two micro-portal VATS would be more accessible for surgeons who are already familiar with the single-direction VATS and could yield the similar minimal invasiveness and less pain as uniport VATS, we believe that the presented two-micro-portal video-assisted thoracic segmentectomy is technically safe and feasible in lung cancer treatment and is worthy of promotion.