Background: Treatment of non-small cell lung cancer (NSCLC) with EGFR exon 20 mutations is an unmet medical need. Poziotinib is a potent tyrosine kinase inhibitor (TKI) of EGFR and HER2 exon 20 insertion mutants. We evaluated the efficacy and safety of poziotinib in previously treated NSCLC patients with EGFR exon 20 insertion mutations in an independent cohort of a multi-cohort, multi-center Phase 2 study (ZENITH20-1). Methods:ZENITH20-1 study enrolled pts with advanced NSCLC with an EGFR exon 20 insertion identified on local tissue testing who had received at least one prior line of therapy. Poziotinib (16 mg) was administered orally QD, allowing dose reductions for AEs, with follow up for 24 months. The primary endpoint was objective response rate (ORR), evaluated centrally by RECIST v1.1. Secondary endpoints included disease control rate (DCR), duration of response (DOR), progression-free survival (PFS) and safety. Efficacy was also evaluated by specific exon 20 insertions and prior lines of therapy. Results: 115 patients with a median age of 61 years (33-83) were enrolled. Patients had a median of 2 prior lines of therapy (range, 1-9.) The median relative dose intensity was 72% (7-100%) with 65% having dose reductions. The ORR in the as-treated population was 14.8% (95% CI 8.9 - 22.6%), and the DCR was 68.7% (95% CI 59.4 - 77.0%) with a median DoR of 7.4 months. 65% patients had tumor size reductions and the median PFS was 4.2 months. In the evaluable population (n = 88), the ORR was 19.3% and the unconfirmed ORR was 25%. Responses were predominantly observed in insertions between residues M766 to D770 of exon 20 (8/44; 18.2%). Responses were observed in patients with 2 lines (14%); ≥3 lines of therapy (16.2%). The most common treatment-related Grade ≥3 AEs were rash (28%), diarrhea (26%), stomatitis (9%) and paronychia (6%). The incidence of treatment-related pneumonitis was 4%, however some cases may have been confounded by prior checkpoint inhibitors as first line treatment. Conclusions: Although the ORR primary endpoint was not met, poziotinib induced tumor reduction in the majority of patients with durable responses, including the heavily pre-treated population. Responses were more common in patients with insertions between M766 to D770 of EGFR exon 20. The safety profile was overall consistent with other 2^nd generation EGFR TKIs. Evaluation of these subgroups with refined dosing and improved toxicity management to maintain continuous treatment is warranted to assess the potential of poziotinib in Exon20 related tumors. Clinical trial information: NCT03318939.