Purpose <p>To evaluate the feasibility and safety of the transfissural route for computed tomography (CT)-guided preoperative localization with medical adhesive for video-assisted thoracoscopic surgery (VATS) resection of challenging pulmonary nodules.</p> Materials and Methods <p>This retrospective study analyzed patients who underwent CT-guided medical adhesive before VATS resection between January 2019 and August 2024. Patients were categorized into transfissural (Group T, <i>n</i> = 31) and conventional (Group C, <i>n</i> = 513) route groups. Propensity score matching (1:4) was performed to control for baseline variables, resulting in 27 matched patients in Group T and 69 in Group C. Localization and surgical outcomes were compared between the two groups. Multiple linear regression analysis was performed to verify the independent association with post-localization pain score.</p> Results <p>In the overall cohort, both groups achieved 100% localization success. No significant differences were observed in initial failure rate, resection success, localization time, or complication rates (including hemorrhage and pneumothorax) between groups, both before and after matching (all <i>P</i> &gt; 0.05). However, Group T was associated with significantly higher pain scores both before (median 4 vs. 3, <i>P</i> &lt; 0.001) and after matching (median 4 vs. 3, <i>P</i> = 0.008), despite comparable morphine use (<i>P</i> = 0.947 post-matching). Multiple linear regression further confirmed the transfissural route as an independent factor significantly associated with increased post-localization pain (<i>P</i> &lt; 0.001).</p> Conclusion <p>The transfissural route for CT-guided medical adhesive localization is a technically feasible and safe strategy for challenging pulmonary nodules. However, the increased pain score should be considered.</p> Level of Evidence 3 <p>Non-controlled retrospective cohort study.</p> Graphical Abstract <p></p>