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Background: Regulatory T-cells (Treg) play key roles in suppressing cell-mediated immunity in cancer patients. Little is known about perioperative Treg fluctuations in nonsmall cell lung cancer (NSCLC). Video-assisted thoracoscopic (VATS) lobectomy, as a minimal invasive procedure for treating NSCLC, may have relatively less impact on the patient′s immune system. This study aimed to observe perioperative dynamics of circulating Treg and natural killer (NK) cell levels in NSCLC patients who underwent major lobectomy by VATS or thoracotomy. Methods: Totally, 98 consecutive patients with stage I NSCLC were recruited and assigned into VATS or thoracotomy groups. Peripheral blood samples were taken on 1-day prior to operation, postoperative days (PODs) 1, 3, 7, 30, and 90. Circulating Treg and NK cell counts were assayed by flow cytometry, defined as CD4 + CD25 + CD127 low cells in CD4 + lymphocytes and CD56 + 16 + CD3− cells within CD45 + leukocytes respectively. With SPSS software version 21.0 (SPSS Inc., USA), differences between VATS and thoracotomy groups were determined by one-way analysis of variance (ANOVA), and differences between preoperative baseline and PODs in each group were evaluated by one-way ANOVA Dunnett t-test. Results: In both groups, postoperative Treg percentages were lower than preoperative status. No statistical difference was found between VATS and thoracotomy groups on PODs 1, 3, 7, and 30. On POD 90, Treg percentage in VATS group was significantly lower than in thoracotomy group (5.26 ± 2.75 vs. 6.99 ± 3.60, P = 0.012). However, a higher level of NK was found on all PODs except on POD 90 in VATS group, comparing to thoracotomy group. Conclusions: Lower Treg level on POD 90 and higher NK levels on PODs 1, 3, 7, 30 in VATS group might imply better preserved cell-mediated immune function in NSCLC patients, than those in thoracotomy group.