Objectives Routine imaging modalities combined with state-of-the-art reconstruction software can substantially improve preoperative planning and simplify complex procedure by enhancing the surgeon’s knowledge of the patient’s specific anatomy. The aim of the current study was to demonstrate the feasibility of interactive three-dimensional (3D) computed tomography (CT) reconstructions for preoperative planning and intraoperative guiding in video-assisted thoracoscopic lung surgery (VATS) with 3D vision. Methods Twenty-five consecutive patients referred for an anatomic pulmonary resection by a single surgeon were included. Data were collected prospectively. All patients underwent a CT angiography in the diagnostic pathway prior to referral. 3D reconstruction of the pulmonary anatomy was obtained from CT scans with dedicated software. An interactive PDF file of the 3D reconstruction with virtual resection was created, in which all the pulmonary structures could be individually selected. Furthermore, the reconstructions were used for intraoperative guiding on double monitor during VATS with 3D vision. Results In total, 26 procedures were performed for 5 benign and 21 malignant conditions. Lobectomy and segmentectomy were performed in 20 (76.9 %) and 6 (23.1%) cases, respectively. In all patients, preoperative 3D reconstruction of pulmonary vessels corresponded with the intraoperative findings. Reconstructions revealed anatomic variations in 4 (15.4%) patients. No conversion to thoracotomy or in-hospital mortality occurred. Conclusions Preoperative planning with interactive 3D CT reconstruction is a useful method to enhance the surgeon’s knowledge of the patient’s specific anatomy and to reveal anatomic variations. Intraoperative 3D guiding in VATS with 3D vision is feasible and could contribute to the safety and accuracy of anatomic resection.