Infiltration of regulatory T (Treg) cells into many tumor types correlates with poor patient prognoses. However, mechanisms of intratumoral Treg cell function remain to be elucidated. We investigated Treg cell function in a genetically engineered mouse model of lung adenocarcinoma and found that Treg cells suppressed anti-tumor responses in tumor-associated tertiary lymphoid structures (TA-TLSs). TA-TLSs have been described in human lung cancers, but their function remains to be determined. TLSs in this model were spatially associated with >90% of tumors and facilitated interactions between T cells and tumor-antigen-presenting dendritic cells (DCs). Costimulatory ligand expression by DCs and T cell proliferation rates increased in TA-TLSs upon Treg cell depletion, leading to tumor destruction. Thus, we propose that Treg cells in TA-TLSs can inhibit endogenous immune responses against tumors, and targeting these cells might provide therapeutic benefit for cancer patients. Display omitted •TA-TLSs form adjacent to advanced lung tumors•TA-TLSs have features and functions of lymph nodes•Treg cells in TA-TLSs actively suppress immune responses•Therapeutic Treg cell depletion causes immune-mediated tumor destruction Intratumoral regulatory T (Treg) cells are important in lung cancer, but it has been difficult to dissect their mechanisms of action. Jacks and colleagues demonstrate that intratumoral Treg cells function within tumor-associated tertiary lymphoid structures to suppress anti-tumor T cell responses in a mouse model of lung cancer.