Background: Cross-signaling in the ErbB family is an important mechanism in Trastuzumab resistance. Pyrotinib is an irreversible pan-ErbB inhibitor targeting EGFR/HER1, HER2 and HER4, which may offer the potential for improved efficacy to block HER2 signaling in trastuzumab-resistant breast cancer. This phase I study assessed the safety, tolerability, maximum-tolerated dose (MTD), pharmacokinetics, antitumor activity and predictive biomarkers of pyrotinib in combination with capecitabine in patients with HER2-positive metastatic breast cancer (MBC). Methods: Patients received oral pyrotinib 160 mg, 240 mg, 320 mg, or 400 mg once daily continually plus capecitabine 1000mg/m² twice daily on days 1 to 14 of a 21-day cycle. Pharmacokinetic blood samples were collected predose on day 1 and day 14 of treatment. Next-generation sequencing (NGS) was performed on circulating tumor DNA (ctDNA) to probe for predictive biomarkers of this combination. Results: A total of 28 patients were enrolled. All 28 (100%) patients experienced at least one treatment-related Adverse Events (AE), which were predominantly grade 1 or 2. Grade 3 treatment-related AE occurred in 12 (42.9%) patients; anemia (14.3%) and diarrhea (10.7%) were the most common grade 3 AEs. Three (10.7%) patients discontinued capecitabine administration due to AEs. The overall response rate (ORR) was 78.6% (95% CI: 59.0% to 91.7%), and the disease control rate (complete response+ partial response+ stable disease) was 96.4% (95% CI: 81.7% to 99.9%). The median progression-free survival (PFS) was 22.1 months (95% CI: 9.0 to 26.2 months). ORR was 70.6% (12/17) in trastuzumab-pretreated patients and 90.9% (10/11) in trastuzumab-naive patients. NGS analysis of all genetic alterations of HER2 bypass signaling pathway, PI3K/Akt/mTOR pathway and TP53 in baseline blood samples suggested that concomitant (two or more) genetic alterations were significantly associated with poorer PFS compared to none or one genetic alteration (median, 15.8 vs. 26.2 months, p = 0.006). Conclusions: Pyrotinib in combination with capecitabine are well-tolerated and demonstrate promising antitumor activity in HER2-positive MBC patients. Clinical trial information: NCT02361112