Human epidermal growth factor receptor 2 (HER2) inhibitors such as trastuzumab and lapatinib are used for breast cancer or gastric cancer that tests HER2 positive. However, as with other targeted therapies, the occurrence of intrinsic or acquired resistance to HER2 inhibitors such as trastuzumab and lapatinib is an unresolved therapeutic problem for HER2-positive gastric cancer. The present study describes how the heat shock protein 90 (HSP90) inhibitor AUY922 could be an alternative treatment for primary lapatinib-resistant (ESO26 and OE33) and lapatinib-sensitive gastric cancer cells (OE19, N87, and SNU-216) harboring HER2 amplification. In order to investigate whether AUY922 could overcome intrinsic and acquired resistance to HER2 inhibitors in HER2-positive gastric cancer, we generated lapatinib-resistant gastric cancer cell lines (OE19/LR and N87/LR) by continuous exposure to lapatinib in vitro. We found that activation of HER2 and protein kinase B (AKT) was a key factor in inducing intrinsic and acquired lapatinib-resistant gastric cancer cell lines, and AUY922 effectively suppressed activation of both HER2 and AKT in acquired lapatinib-resistant gastric cancer cell lines. In conclusion, AUY922 shows a synergistic anti-cancer effect with lapatinib and could sensitize gastric cancer cells that have intrinsic resistance to lapatinib. In addition, this dual inhibition of the HSP90 and HER2 signaling pathways could represent a potent therapeutic strategy to treat HER2-positive gastric cancer with intrinsic and acquired resistance to lapatinib.