Background: HER2 exon 20 mutation is a well-known oncogenic driver in non-small cell lung cancer (NSCLC) and its testing is recommended by NCCN guidelines for NSCLC patients. However, up to now, there has been no approved targeted therapy for this patient population. Pyrotinib is an oral, irreversible pan-Her tyrosine kinase inhibitor (TKI) against HER1, HER2 and HER4, which, combined with chemotherapy, has recently been approved in China for HER2 positive advanced breast cancer. Methods: Stage IIIB or IV NSCLC patients with HER2 exon 20 mutation (confirmed in a central lab) and previously treated with at least one platinum-based chemotherapy were enrolled in this open-label, multi-center, single-arm phase II study. Patients with active brain metastases or prior use of HER2 TKI agents were excluded. Eligible patients received pyrotinib 400 mg once daily until disease progression, intolerable toxicity or withdrawal of consent. The primary endpoint was objective response rate (ORR) according to RECIST 1.1. Secondary endpoints included safety, progression-free survival (PFS), time to disease progression (TTP), duration of response (DOR) and overall survival (OS). This trial is registered on ClinicalTrials.gov (NCT02834936). Results: Between October 20, 2016 and December 10, 2018, 60 patients (33 female, 27 male) were enrolled. The median age was 57 years (range: 40, 72). A majority were stage IV patients (95%). Over 58% of patients have been treated with two or more prior chemotherapy regimens. As of Jan 21, 2019, the ORR as evaluated by investigators was 31.7% (95% CI 20.3%, 45.0%). Median DOR was 7.0 months (95% CI 5.5, 11.0) and median PFS was 6.8 months (95% CI 4.1, 8.3). 26.7% patients reported treatment-related grade 3 AEs. Diarrhea (20.0%) was the only treatment-related grade 3 AE that occurred in ≥ 2 patients. Treatment-related grade 4 AE was reported in 1 subject (elevated gamma-glutamyl transferase). The anti-tumor activities of pyrotinib was shown in various mutation types. Conclusions: Pyrotinib as a single agent demonstrated promising anti-tumor activity and acceptable safety profile in heavily pretreated HER2 mutant NSCLC patients. Clinical trial information: NCT02834936