Nucleophosmin (NPM) and human epidermal growth factor receptor-2 (Her-2) are abnormally expressed in various types of human malignant tumors, including gastric cancer, and have been closely associated with cancer chemoresistance. However, their interaction and roles in oxaliplatin resistance are not fully understood. Therefore, the present study aimed to elucidate the relationship between NPM and Her-2 in gastric cancer cell lines and clinical samples, and further investigated their role in the resistance of gastric cancer to oxaliplatin. Western blotting and reverse transcription-quantitative polymerase chain reaction confirmed that NPM and Her-2 expression were significantly upregulated in gastric cancer cells and clinical samples, and that their expression levels were strongly correlated. However, Her-2 expression was not affected by upregulation or downregulation of NPM expression in gastric cancer cells. Cell counting kit-8 assays demonstrated that the cell sensitivity to oxaliplatin decreased simultaneously with an increase in NPM expression. Furthermore, inhibition of Her-2 expression using trastuzumab significantly increased the sensitivity of the cells to oxaliplatin, which occurred simultaneously with the downregulation of NPM. These results indicated that inhibition of NPM, as a Her-2 downstream signal, may be a novel strategy to overcome oxaliplatin-resistant gastric cancer, and that trastuzumab and oxaliplatin may exhibit a synergistic antitumor effect in Her-2-positive gastric cancer cells.