Regulatory T cells (Tregs) suppress antitumor immune responses. Cycles of Dendritic cells (DC) vaccination combined with cytokine-induced killer (CIK) cells (DC/CIK) treatment were significantly related with good prognosis. Therefore, we investigated whether increased cycles of immunotherapy could decrease frequency of Tregs in patients with resected non-small cell lung cancer (NSCLC). Previous study from our laboratory has determined that the optimal cutoff point of the cycle count was 3cycles. We examined the levels of Tregs and the related cytokines by flow cytometric and cytokine analysis in these patients after more than (≥) 3cycles or less than (<) 3cycles of DC/CIK cell treatment. Significant reduction of Tregs frequency, Treg-generated cytokines level and recurrence rate were presented in patients received with ≥3cycles of DC/CIK cell treatment compared with patients with <3cycles of treatment. Interestingly, Tregs frequency and the related cytokines level were similar between patients suffered tumor recurrence and patients without recurrence in both groups. Together, our findings reveal that increased cycle count of DC/CIK cell immunotherapy contribute to decline of Tregs frequency and cancer recurrence rate in patients with resected NSCLC.