Background: Addition of trastuzumab to cisplatin and fluoropyrimidine-based doublet chemotherapy is the standard first-line treatment for patients with human epidermal growth factor receptor 2 (HER2)-positive advanced or recurrent gastric cancer. However, second-line anti-HER2 therapy did not demonstrate a survival advantage. We assessed the change in HER2 status in cases of HER2-positive gastric cancer refractory to trastuzumab, and analyzed potential biomarkers associated with resistance to trastuzumab. Methods: Key inclusion criteria were as follows; advanced or recurrent gastric adenocarcinoma with HER2-positive status (IHC3+ or IHC2+ with FISH amplification) before trastuzumab treatment; radiologically or clinically diagnosed with progressive disease following trastuzumab treatment; pathologically confirmed adenocarcinoma within 3 months following completion of trastuzumab treatment without receipt of any other anti-tumor agents. We collected biopsy samples from patients who had developed resistance to trastuzumab and evaluated HER2 status before and after. Amplification of EGFR and c-met, as well as PIK3CA mutation were also comparatively analyzed if samples were available. Results: Biopsy samples and clinical data were collected from 33 eligible patients as the full analysis set. HER2 loss was identified in 20 patients (60.6%) with refractory disease. IHC showed that HER2 overexpression was remarkably decreased after treatment (pre-HER2 IHC 3+: 24 [72.7%]; 2+: 9 [27.3%] vs. post-HER2 3+: 13 [39.4%]; 2+: 1 [3.0%]; 1+: 18 [54.5%]; 0: 1 [3.0%]). EGFR amplification, c-met amplification, and PIK3CA gene mutation before and after trastuzumab treatment was observed in 7.7% and 4.2%, 15.8% and 6.7%, and 6.3% and 7.1% of cases, respectively. Conclusions: HER2 loss occurred in a considerable number of patients who were initially diagnosed with HER2-positive gastric cancer, resulting in resistance to trastuzumab. EGFR, c-met and PIK3CA were rarely associated with acquired resistance. Re-evaluation of HER2 status might provide valuable clues with regard to anti-HER2 targeted therapy beyond disease progression.