Although there are effective HER2-targeted agents, novel combination strategies in HER2-overexpressing breast cancers are needed for patients whose tumors develop drug resistance. To develop new therapeutic strategy, we investigated the combinational effect of entinostat, an oral isoform-selective histone deacetylase type I inhibitor, and lapatinib, a HER2/EGFR dual tyrosine kinase inhibitor, in HER2+ breast cancer cells. We assessed the combinational synergistic effect and its mechanism by CellTiter Blue assay, flow cytometry, anchorage-independent growth, quantitative real-time PCR, small interfering RNA, Western blotting, and mammary fat pad xenograft mouse models. We found that compared with entinostat or lapatinib alone, the two drugs in combination synergistically inhibited proliferation (P < 0.001), reduced in vitro colony formation (P < 0.05), and resulted in significant in vivo tumor shrinkage or growth inhibition in two xenograft mouse models (BT474 and SUM190, P < 0.001). The synergistic anti-tumor activity of the entinostat/lapatinib combination was due to downregulation of phosphorylated Akt, which activated transcriptional activity of FOXO3, resulting in induction of Bim1 (a BH3 domain-containing pro-apoptotic protein). Furthermore, entinostat sensitized trastuzumab/lapatinib-resistance-HER2-overexpressing cells to the trastuzumab/lapatinib combination and enhanced the anti-proliferation effect compare with single or double combination treatment. This study provides evidence that entinostat has enhanced anti-tumor effect in combination with HER2-targeted reagent, lapatinib, and resulting in induction of apoptosis by FOXO3-mediated Bim1 expression. Our finding justifies for conducting a clinical trial of combinational treatment with entinostat, lapatinib, and trastuzumab in patients with HER2-overexpressing breast cancer resistant to trastuzumab-based treatment.