Human epidermal growth factor receptor 2 (HER2) targeted therapy is currently considered as the standard treatment for HER2-positive advanced gastric cancer (GC). However, unsatisfactory results of recent phase III clinical trials involving lapatinib suggested biomarkers for selection of patients. The aim of this study was to identify JWA as a biomarker for lapatinib resistance in GC cells and elucidate the underlying mechanisms. Lapatinib was effective to the intrinsic cisplatin-resistant GC cells. JWA activation conferred lapatinib unresponsiveness, but reversed cisplatin resistance in GC cells. Whereas, deletion of JWA significantly restored lapatinib suppression on proliferation and lapatinib-induced apoptosis. JWA-induced down-regulation of HER2 and activation of ERK phosphorylation led to lapatinib resistance. Furthermore, c-Cbl represented a novel mechanism for HER2 degradation enhanced by JWA in GC cells. Taken together, JWA is a potential predictive marker for lapatinib resistance, targeting the patients that may benefit from lapatinib treatment in human GC.