Background A minority of patients with advanced non-small-cell lung cancer (NSCLC) benefit from treatment with immune checkpoint inhibitors (ICIs). Ineffective effector function of activated T and NK cells may lead to reduced tumor cell death, even when these activated effector cells are released from their immune checkpoint brake. Hence, in this study we aimed to assess the association of baseline serum granzyme B, as well as germline variation of the GZMB gene, with clinical outcome to programmed cell death protein 1 (PD-1) blockade. Methods A total of 347 patients with stage IV NSCLC who started nivolumab treatment between June 2013 and June 2017 were prospectively included. Baseline serum and whole blood was available, allowing for protein quantification and targeted DNA sequencing. Clinical outcome was based on best overall response (BOR) according to Response Evaluation Criteria in Solid Tumors, V.1.1, progression-free survival (PFS), and overall survival (OS). Results Patients with low serum levels of granzyme B had worse PFS (HR: 1.96; 95% CI: 1.12 to 3.43; p=0.018) and worse OS (HR: 2.08; 95% CI: 1.12 to 3.87; p=0.021) than patients with high baseline serum levels. To validate the findings, germline variation of GZMB rs8192917 was assessed. Patients with homozygous and heterozygous variants of GZMB rs8192917 had worse BOR (OR: 1.60; 95% CI: 1.01 to 2.52; p=0.044) and worse PFS (HR: 1.38; 95% CI:1.02 to 1.87; p=0.036) than wild types. Conclusions A low baseline serum level of granzyme B and germline variation of GZMB was associated with worse clinical outcome in NSCLC, emphasizing the relevance and additional value of monitoring germline genetic variations which mirror cytotoxic functions of T cells in ICI therapy. Trail registration number Dutch Trial Registry (NL6828).