BACKGROUND: The combination of epirubicin, cisplatin, and 5-fluorouracil (ECF) is widely used for gastric cancer treatment. However, cancer cells can acquire chemoresistance over multiple treatment cycles, leading to recurrence. This study aimed to investigate a novel biomarker for predicting ECF resistance and its biological roles in gastric cancer. METHODS: ECF-resistant (ECF-R) gastric cancer cell lines were established through stepwise ECF treatment. Transcriptome analysis was performed to identify resistance-related genes, which were validated in tumor organoids and in vivo models. Additionally, gastric cancer patient tumor tissues were analyzed for clinical relevance. RESULTS: Transcriptome analysis revealed that NINJURIN2 and CD44 were highly expressed in ECF-R cells but rarely expressed in normal gastric tissues. NINJURIN2 inhibition significantly increased chemosensitivity to ECF in vitro and in vivo. Liquid chromatography-tandem mass spectrometry identified periostin as a binding partner of NINJURIN2, mediating chemoresistance. Furthermore, VAV2 phosphorylation was markedly upregulated in ECF-R cells but was inhibited by NINJURIN2 knockdown. Clinical analysis showed that high NINJURIN2 expression correlated with poor survival outcomes in gastric cancer patients. CONCLUSION: Our findings suggest that NINJURIN2 can be used as a novel biomarker for chemoresistant gastric cancer patients and that inhibiting NINJURIN2 along with standard chemotherapy could prevent chemoresistance-associated relapse in gastric cancer.