LAG3 is a surface molecule found on a subset of immune cells. The precise function of LAG3 appears to be context-dependent. In this study, we investigated the effect of LAG3 on CD4+CD25- T cells from non-small cell lung cancer (NSCLC) patients. We found that in the peripheral blood mononuclear cells of NSCLC patients, LAG3 was significantly increased in CD4+ T cells directly ex vivo and primarily in the CD4+CD25- fraction, which was regulated by prolonged TCR stimulation and the presence of IL-27. TCR stimulation also increased CD25 expression, but not Foxp3 expression, in LAG3-expressing CD4+CD25- cells Compared to LAG3-nonexpressing CD4+CD25- cells, LAG3-expressing CD4+CD25- cells presented significantly higher levels of PD1 and TIM3, two inhibitory receptors best described in exhausted CD8+ T effector cells. LAG3-expressing CD4+CD25- cells also presented impaired proliferation compared with LAG3-nonexpressing CD4+CD25- cells but could be partially rescued by inhibiting both PD1 and TIM3. Interestingly, CD8+ T cells co-incubated with LAG3-expressing CD4+CD25- cells at equal cell numbers demonstrated significantly lower proliferation than CD8+ T cells incubated alone. Co-culture with CD8+ T cell and LAG3-expressing CD4+CD25- T cell also upregulated soluble IL-10 level in the supernatant, of which the concentration was positively correlated with the number of LAG3-expressing CD4+CD25- T cells. In addition, we found that LAG3-expressing CD4+CD25- T cells infiltrated the resected tumors and were present at higher frequencies of in metastases than in primary tumors. Taken together, these data suggest that LAG3-expressing CD4+CD25- T cells represent another regulatory immune cell type with potential to interfere with anti-tumor immunity.