Objective To evaluate the effectiveness and safety of single-incision video-assisted thoracic surgery versus conventional multiple ports video-assisted thoracic surgery in the treatment of lung cancer as well as providing reference for clinical decision-making. Methods We searched the Cochrane Library, PubMed, EMbase, CBM, CNKI, VIP, Wanfang ect until March 2017 to collect randomized controlled trials (RCTs), cohort studies, and case-control studies comparing single-incision with conventional multiple ports video-assisted thoracic surgery for lung cancer.Two reviewers independently screened and selected literatures according to inclusion and exclusion criteria. Then data extraction and quality assessment of included studies were conducted. RevMan 5.3 software was used for meta-analysis. Results Twenty-six cohort studies (3 053 patients) were included. The quality of the included studies was high with score more than five.Meta-analysis showed that single-incision video-assisted thoracic surgery had shorter thoracic drainage time (MD=–0.71, 95% CI –1.03 to –0.39), shorter hospitalization time (MD=–0.92, 95% CI –1.66 to –0.19), lower pain scores 1 day after surgery (MD=–0.65, 95% CI –0.90 to –0.40), lower pain scores 3 days after surgery (MD=–0.90, 95% CI –1.16 to –0.64), lower pain scores 7 days after surgery (MD=–1.24, 95% CI –1.90 to –0.57), less number of lymph node dissection (MD=–0.72, 95% CI –1.35 to –0.10), less total drainage fluid (MD=–108.60, 95% CI –180.42 to –36.79) and shorter length of surgical incision (MD=–2.74, 95% CI –3.57 to –1.90) than conventional multiple ports video-assisted thoracic surgery. But the differences between the two groups in operation time, intraoperative blood loss, postoperative complications were not statistically significant. Conclusion Single-incision video-assisted thoracic surgery is safer and better in patient's compliance than conventional multiple ports video-assisted thoracic surgery in the treatment of lung cancer. But there is no significant difference in operation time, intraoperative blood loss, or postoperative complications. It still needs large-scale, high-quality studies to demonstrate its effectiveness and safety.