<jats:title>Abstract</jats:title> <jats:p>Killer cell lectin-like receptor G1 (KLRG1) is a co-inhibitory receptor expressed on T and NK cells. While KLRG1’s role in effector T and NK cells has been extensively documented, its impact on regulatory T cells (Tregs) remains poorly understood. This study aims to characterize and compare the immunosuppressive potential of KLRG1+ Tregs in contrast to KLRG1- Tregs and their therapeutic targeting in non-small cell lung cancer (NSCLC).</jats:p> <jats:p>CTV-labelled effector T cells were cultured with TAMs and tumor digests in the presence or absence of each Treg subpopulation for 5 days in a Treg suppression assay. For in vivo studies, T cells with either KLRG1+ or KLRG1- Tregs were adoptively transferred into lung tumor-bearing mice. Intracellular staining for GZB, IFN-γ and TNF-α was performed on the processed tumor cells. Lastly, cohorts of tumor-bearing KP mice were treated with a KLRG1-blocking antibody for 6 weeks.</jats:p> <jats:p>Our study revealed that KLRG1+ Tregs exhibit robust suppressive functions both in vitro and in vivo, contingent upon intact KLRG1 signaling. Blockade of KLRG1 in vitro and in vivo profoundly arrested tumor growth, augmented effector T cell activity, and significantly prolonged the survival of tumor-bearing mice.</jats:p> <jats:p>In conclusion, KLRG1+ Tregs drives immunosuppressive activity in the tumor microenvironment; this provides novel insights into the functional dynamics of KLRG1+ Tregs and underscores their therapeutic potential for boosting anti-tumor T cell responses in NSCLC.</jats:p>