Objective To evaluate the effectiveness and safety of pure carbon dioxide (CO2) combined with a modified inflation-deflation technique for identifying the intersegmental plane during thoracoscopic segmentectomy. Methods A prospective study was conducted, enrolling 30 patients diagnosed with pulmonary nodules who underwent thoracoscopic anatomical segmentectomy at the Department of Thoracic Surgery, the First Affiliated Hospital of Xi'an Jiaotong University, from March 2024 to March 2025. Patients were randomly assigned to one of two groups using a random number table: A pure oxygen group (O2 group, n=15, 8 females, 7 males, age 28-75 years) and a pure carbon dioxide group (CO2 group, n=15, 8 females, 7 males, age 37-69 years). All patients underwent preoperative three-dimensional computed tomography bronchovascular angiography to reconstruct pulmonary vessels, bronchi, and the virtual intersegmental plane. The time to identification of the ideal intersegmental plane was recorded intraoperatively, along with arterial blood gas measurements before lung inflation and at 5 and 15 minutes after lung inflation on the surgical side. Results The time to identify the intersegmental plane was significantly shorter in the CO2 group compared to the O2 group [(151.1±39.5) s vs. (998.7±78.9) s, P<0.001], and there were no significant fluctuations in intraoperative oxygen saturation in patients in the CO2 group. Furthermore, there were no statistically significant differences between the two groups in terms of operation duration, intraoperative blood loss, postoperative extubation time, total postoperative chest tube drainage, postoperative length of hospital stay, or postoperative complication rate (all P>0.05). Conclusion Pure CO2 combined with a modified inflation-deflation technique can rapidly, accurately, and clearly identify the intersegmental plane, and its safety is non-inferior to that of the pure O2 method, making it worthy of clinical promotion and application.