HER2 amplification is found in more than 15% of gastric cancers and is associated with poor clinical outcome. Lapatinib, a dual HER2 and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has shown promising in vitro results in treating HER2⁺ cancer cells. However, several studies have shown that activation of alternative receptor tyrosine kinases can mediate resistance to HER-targeted therapy. Here, we investigated whether activated MET can confer resistance to lapatinib inhibition of gastric cancer cells. A panel of gastric cancer cell lines was treated with lapatinib, and we observed that cell proliferation was reduced by 70% and that the degree of HER2 amplification corresponds to sensitivity to lapatinib. Immunoblotting analysis indicated that phosphorylation of HER2, EGFR, MET, AKT, and extracellular signal-regulated kinase was inhibited by lapatinib and presumably led to cell-cycle arrest as observed with flow cytometry. Hepatocyte growth factor (HGF) activation of MET receptors rescued cells from lapatinib-induced growth inhibition by restimulating the downstream pathways and restoring normal cell-cycle progression. This rescue effect could be abrogated by inhibiting MET with PHA-665752 (a highly specific MET inhibitor) or downregulating MET expression with short interfering RNA. No synergy in growth inhibition was observed when cells were treated with a combination of lapatinib and PHA-665752. Repeat studies using insulin-like growth factor 1 and fibroblast growth factor 3 could not uniformly rescue the lapatinib-treated gastric cancer cells. In conclusion, HGF/MET–mediated resistance to lapatinib is a novel mechanism of resistance to HER2-targeted agents in gastric cancer cells. Development of inhibitors targeting multiple receptors or common downstream signaling proteins merits further investigation. Mol Cancer Ther; 11(3); 660–9. ©2012 AACR.