We report a phase II study of 50 advanced non-small cell lung cancer (NSCLC) patients with point mutations or insertions in EGFR exon 20 treated with poziotinib (NCT03066206). The study achieved its primary endpoint, with confirmed objective response rates (ORRs) of 32% and 31% by investigator and blinded independent review, respectively, with a median progression-free survival of 5.5 months. Using preclinical studies, in silico modeling, and molecular dynamics simulations, we found that poziotinib sensitivity was highly dependent on the insertion location, with near-loop insertions (amino acids A767 to P772) being more sensitive than far-loop insertions, an observation confirmed clinically with ORRs of 46% and 0% observed in near versus far-loop, respectively (p = 0.0015). Putative mechanisms of acquired resistance included EGFR T790M, MET amplifications, and epithelial-to-mesenchymal transition (EMT). Our data demonstrate that poziotinib is active in EGFR exon 20-mutant NSCLC, although this activity is influenced by insertion location. Display omitted •Poziotinib yields a 32% response rate in EGFR exon 20-mutant NSCLC•Poziotinib sensitivity is highly dependent on insertion location•Near-loop exon 20 insertions are more sensitive to poziotinib than far-loop insertions•Mechanisms of acquired poziotinib resistance include EGFR T790M and MET amplifications Elamin et al. show that poziotinib is active in EGFR exon 20-mutant non-small cell lung cancer. The activity of poziotinib is influenced by insertion location in exon 20, with near-loop insertion being more sensitive than far-loop insertion. Poziotinib acquired resistance is mediated via EGFR-dependent and -independent mechanisms.