Abstract Purpose: To investigate targeting of indoleamine 2,3 dioxygenase (IDO) enzyme using a synthetic peptide vaccine administered to patients with metastatic non–small cell lung cancer (NSCLC). Experimental Design: In a clinical phase I study, we treated 15 HLA-A2–positive patients with stage III–IV NSCLC in disease stabilization after standard chemotherapy. Patients were treated with imiquimod ointment and subcutaneous vaccinations (100 μg IDO5 peptide, sequence ALLEIASCL, formulated in 900 μL Montanide). Primary endpoint was toxicity. Clinical benefit and immunity were assessed as secondary endpoints. Results: No severe toxicity was observed. One patient developed a partial response (PR) after one year of vaccine treatment, whereas long-lasting stable disease (SD) ≥ 8.5 months was demonstrated in another six patients. The median overall survival (OS) was 25.9 months. Patients demonstrated significant improved OS (P = 0.03) when compared with the group of patients excluded because of HLA-A2 negativity. IDO-specific CD8+ T-cell immunity was demonstrated by IFN-γ Elispot and Tetramer staining. Fluorescence-activated cell sorting analyses demonstrated a significant reduction of the Treg population (P = 0.03) after the sixth vaccine (2.5 months) compared with pretreatment levels. Furthermore, expression of IDO was detected in nine of ten tumor biopsies by immunohistochemistry. High-performance liquid chromatography analyses of kynurenine/tryptophan (Kyn/Trp) ratio in sera were performed. In long-term analyses of two clinical responding patients, the ratio of Kyn/Trp remained stable. Conclusions: The vaccine was well tolerated with no severe toxicity occurring. A median OS of 25.9 months was demonstrated and long-lasting PR+SD was seen in 47% of the patients. Clin Cancer Res; 20(1); 221–32. ©2013 AACR.