Trastuzumab is the only target to be approved as the first-line treatment of HER2 positive metastatic gastric cancer, but ubiquitous resistance decreases its therapeutic benefit. In this study, we found HER4, phosphorylation HER4 (p-HER4) and the mesenchymal marker Vimentin increased in trastuzumab-resistant cells (MKN45TR and NCI-N87TR), while epithelial markers expressions in trastuzumab-resistant cell lines and animal models decreased. Additionally, silencing HER4 prevented the epithelial-mesenchymal transition and led to decreased proliferation and migration in vitro and in vivo. The expression of YAP1, a vital downstream interacted target of HER4, decreased when HER4 was knocked down. Interestingly, stimulation of NRG1 could compromise the inhibitory impact and rescue cell survival; whereas, transfection of siYAP1 sensitized trastuzumab-treated cells. Expression analysis of the proteins in patient-derived xenograft model (PDX) mice showed that HER4, p-HER4, YAP1, and Vimentin were clearly upregulated in the trastuzumab-resistant mice compared to mice without trastuzumab resistance. However, HER2 and E-cadherin were downregulated in response to continuous treatment with trastuzumab. These findings elucidated that the central role of the HER4-YAP1 axis in trastuzumab resistance of HER2-positive gastric cancer cells through induction of EMT. Hence, regulating the HER4-YAP1 axis might be a promising strategy for clinical interventions in patients with HER2-positive gastric cancer.