Objective: To investigate the effect of down-regulating G protein signal regulating protein 2 (GPSM2) expression on chemotherapy sensitivity of pancreatic cancer cells. Methods: The pancreatic cancer MIA-PaCa-2 cells with low GPSM2 expression were constructed and identified. Sixteen nude mice were equally randomized into two groups, and were subcutaneously implanted with MIA-PaCa-2 cells with low GPSM2 expression and MIA-PaCa-2 cells with natural GPSM2 expression to establish the tumor-bearing models. After that, half mice underwent gemcitabine injection (100 mg/kg) and half mice were given saline of the same volume in each group (intraperitoneal injection, 3 times per week for 4 weeks). The tumor growth curves were drawn, and all mice were sacrificed on the third day after the last injection, and the volumes of the tumor xenografts were determined. Results: The pancreatic cancer MIA-PaCa-2 cells with low GPSM2 expression were successfully created. Both growth speed and volume of the tumor xenografts presented a decreasing trend as natural GPSM2 expression plus saline group>natural GPSM2 expression plus gemcitabine group>low GPSM2 expression plus saline group>low GPSM2 expression plus gemcitabine group. The result of factorial design showed that the main effect of either gemcitabine alone or down-regulating GPSM2 gene expression alone on tumor growth inhibition had statistical significance (both P=0.000), while the interaction effect of gemcitabine combined with down-regulating GPSM2 gene expression on tumor growth inhibition did not reach a statistical significance (P=0.073), but the simple effect of gemcitabine combined with down-regulating GPSM2 gene expression on tumor growth inhibition had statistical significance compared with gemcitabine alone or down-regulating GPSM2 gene expression alone (P=0.000 and 0.003). Conclusion: Whether down-regulating GPSM2 gene expression will enhance chemotherapy sensitivity of pancreatic cancer cells cannot be confirmed. However, the inhibitory effect of down-regulating GPSM2 gene expression with simultaneous chemotherapy on pancreatic cancer is remarkably greater than that of their single actions.