12000 Background: Pain is the most common symptom experienced by patients with advanced cancer. Acetaminophen (APAP, commonly known as paracetamol) alone or in combination with a weak opioid, such as codeine or tramadol, is usually considered as the first-line strategy to manage mild-to-moderate pain in this setting. Although generally considered to be safe, several evidences suggest that APAP may have negative immunomodulatory effects. Randomized studies have shown that APAP use is associated with blunted vaccine immune responses. Given its potential to impair vaccine effectiveness, the World Health Organization stated in 2015 that administration of APAP before or at the time of vaccination is not recommended. This study aimed to assess APAP impact on efficacy of immunotherapy in patients with cancer. Methods: Exposure to APAP was assessed by plasma analysis and was correlated with clinical outcome in three independent cohorts of patients with advanced cancer who were treated with immune checkpoint blockers (ICB): the CheckMate 025 trial, n = 297 (NCT01668784, sponsor: Bristol Myers Squibb), the institutional biomarker program BIP n = 34 (NCT02534649, sponsor: Institut Bergonié, Bordeaux, France) and the institutional biomarker program PREMIS n = 297 (NCT03984318, sponsor: Gustave Roussy, Villejuif, France). APAP immunomodulatory effects were evaluated on a pre-clinical tumor model (MC38) and on human peripheral blood mononuclear cells (PBMCs) from healthy donors. Results: Detectable plasma APAP levels at treatment onset was associated with a significantly worse clinical outcome in ICB-treated cancer patients (HR for progression-free survival : 1.43, 95% CI 1.07–1.91, p = 0.015; HR for overall survival: 1.78 95% CI 1.18–2.68, p = 0.006), independently of other prognostic factors (age, performance status, number of previous lines of treatment, tumor type, number of metastatic sites, presence of liver metastases, LDH levels). APAP significantly reduced ICB efficacy in the pre-clinical MC38 model, as well as the production of PD1 blockade-related interferon-γ secretion by human PBMCs. Moreover, reduction of ICB efficacy in vivo was associated with significantly increased tumor infiltration by regulatory T cells (Tregs). Administration of APAP over 24 h induced a significant expansion of peripheral Tregs in healthy individuals. In addition, interleukin-10, a crucial mediator of Treg-induced immune suppression, was significantly upregulated upon treatment with ICB in cancer patients taking APAP. Conclusions: This study provides strong pre-clinical and clinical evidence of the role of APAP as a potential suppressor of antitumor immunity. Hence, APAP should be used with caution in patients treated with ICB.