Objective To explore the expression and clinical significance of lymphocyte subsets distribution, NK cell activating receptors and regulatory T cells (Tregs) in the peripheral blood of patients with non-small cell lung cancer (NSCLC). Methods A total of 42 NSCLC patients were selected and divided into stage I+II group and stage III+IV group according to tumor stage, or well-differentiated group, moderately-differentiated group and poorly-differentiated group according to the degree of differentiation. Noncancerous lesion group and healthy control group were also set up. Flow cytometry was used to detect the expression levels of lymphocyte subsets, NK cell cytotoxic receptor 3 (NCR3/NKp30), NCR1/NKp46 and Tregs in the peripheral blood. Results Compared with the healthy control group, the proportion of CD3+ T cells significantly increased in the stage III+IV group, the proportion of CD3+CD4+ T cells also significantly increased in the stage III+IV group and the well-differentiated group, and the proportion of Treg in CD4+ T cells went up in all stage groups and poorly-differentiated group. The proportion of NK cells was significantly up-regulated in the stage I+II group and the moderately-differentiated group. The expression of NKp30 and NKp46 on the surface of NK cells was significantly reduced in all stage groups, moderately- and poorly-differentiated groups. Conclusion The proportions of T cells, Tregs and NK cells increase, but the levels of activating receptors on NK cells decrease in the peripheral blood of patients with NSCLC.