Signal transducer and activator of transcription (STAT) 5, including STAT 5a and STAT 5b, was reported to play important roles in the malignant biological behaviors of tumors. However, their roles in gastric cancer, especially for STAT 5b remain unknown. This study aimed to detect the expression of STAT 5b in gastric cancer cells and analyze the role and possible mechanism of STAT 5b in the chemo-sensitivity of gastric cancer cells to gefitinib. A total of 69 patients with gastric carcinomas were analyzed for the expression of STAT 5b in carcinomas and para-carcinomas by immunohistochemistry. Cultured MGC-803 and MKN-45 cells were exposed to gefitinib and/or STAT 5b siRNA. Mitochondrial proteins including Bcl-2, Bax, caspase-3 and caspase-9 were extracted using special kits for detecting mitochondrial pathway-related apoptosis proteins. The results showed that STAT 5b expression was significantly increased in gastric carcinomas compared with para-carcinomas, with a positive rate of 49/69 in carcinomas and 27/69 in para-carcinomas (P=0.001). Gefitinib exposure reduced the relative viabilities of MGC-803 and MKN-45 cells in a concentration- and time-dependent manner, and cell apoptosis increased significantly (P<0.05) with gefitinib treatment (4 mM, 24 h). STAT 5b expression was significantly downregulated by treatment with gefitinib (4 mM, 24 h). Interference of STAT 5b expression by siRNA targeting enhanced the chemo-sensitivity of gastric cancer cells to gefitinib by promoting mitochondrial pathway-mediated apoptosis. Bax, caspase-3 and caspase-9 expression were upregulated, and Bcl-2 expression was downregulated in the combined treatment group (gefitinib+siRNA) compared with the gefitinib (4 mM, 24 h) only group in the MGC-803 and MKN-45 cells (P<0.05). Overall, STAT 5b was upregulated in gastric carcinomas compared with para-carcinomas. Interference of STAT 5b expression by siRNA targeting enhanced the chemo-sensitivity of gastric cancer cells to gefitinib by promoting mitochondrial pathway-mediated cell apoptosis. These findings may be useful for developing new approaches for the treatment of gastric cancer.