Human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC) has been the most challenging subtype of BC, consisting of 20% of BC with an apparent correlation with poor prognosis. Despite that pyrotinib, a new HER2 inhibitor, has led to dramatic improvements in prognosis, the efficacy of pyrotinib monotherapy remains largely restricted due to its acquired resistance. Therefore, identifying a new potential antitumor drug in combination with pyrotinib to amplify therapeutic efficacy is a pressing necessity. Here, we reported a novel combination of pyrotinib with chrysin and explored its antitumor efficacy and the underlying mechanism in HER2-positive BC. We determined that pyrotinib combined with chrysin yielded a potent synergistic effect to induce more evident cell cycle arrest, inhibit the proliferation of BT-474 and SK-BR-3 BC cells, and repress in vivo tumor growth in xenograft mice models. This may be attributed to enhanced autophagy induced by endoplasmic reticulum stress. Furthermore, the combined treatment of pyrotinib and chrysin induced ubiquitination and glucose-6-phosphate dehydrogenase (G6PD) degradation by upregulating zinc finger and BTB/POZ domain-containing family protein 16 (ZBTB16) in tumorigenesis of BC. Mechanistically, we identified that miR-16-5p was a potential upstream regulator of ZBTB16, and it showed a significant inverse correlation with ZBTB16. Inhibition of miR-16-5p overexpression by restoring ZBTB16 significantly potentiated the overall antitumor efficacy of pyrotinib combined with chrysin against HER2-positive BC. Together, these findings demonstrate that the combined treatment of pyrotinib and chrysin enhances autophagy in HER2-positive BC through an unrecognized miR-16-5p/ZBTB16/G6PD axis.© 2023. The Author(s).