The aim of the present study was to determine whether gastric cancer chemoresistance was increased under high glucose conditions by means of a clinical case study and experimental cytology. The expression of nicotinamide phosphoribosyltransferase (Nampt), silent information regulator 1 (sirt1), p53, p-glycoprotein (P-gp) and topoisomerase (topo)-IIα was evaluated in gastric cancer tissues and gastric cancer with diabetes tissues by immunohistochemistry. Subsequently, the survival time of the patients was assessed. For further investigation, the human gastric cancer cell line SGC7901 was subjected to different glucose concentrations and the aforementioned proteins were detected using reverse transcription-quantitative polymerase chain reaction and western blot analysis. Finally, cell sensitivity to chemotherapy treatment was examined in order to elucidate the role of high glucose in MDR. Positive expression of Nampt, Sirt1, p53, P-gp and Topo-IIα was observed to be higher in gastric cancer with diabetes patients compared with gastric cancer patients (P=0.01, 0.003, 0.0025, 0.016 and 0.336, respectively) with reduced survival time. Similar results were observed in SGC7901 cells. Additionally, cell proliferation rates of SGC7901 cells increased at glucose concentrations of 4,500 and 9,000 mg/l. Notably, the inhibition rates of 5-fluorouracil on cells decreased over 48 h when treated with 4,500 and 9,000 mg/l glucose compared with 1,000 mg/l. In conclusion, patients suffering from gastric cancer and diabetes exhibited greater negative effects, such as a poorer response to chemotherapy and had a lower survival time. High glucose conditions promoted gastric cancer cell proliferation and reduced susceptibility to chemotherapy drugs. These data provided a potential diagnostic and therapeutic strategy for gastric cancer chemoresistance.