The aim of the present study was to determine the effect of AICAR, an AMPK activator, on apoptosis in gastric carcinoma cells (SGC-7901) with or without 5-fluorouracil (5-FU). SGC-7901 cells were treated with AICAR (0.2-5 mM, for 24-48 h) with or without 5-FU. Cell viability was determined using MTT assay, while apoptosis were measured through the evaluation of active caspase-3 activity and DNA fragmentation. Real-time PCR was employed to determine the expression of tumor suppressor and multi-drug resistant (mdr1) gene. Cleaved caspase-3 and phosphorylated AMPK (p-AMPK) were measured by Western blot. AICAR significant reduced cellular viability but increased apoptosis in a time- and dose-dependent manner, which is associated with an increase in p-AMPK levels. Importantly, AICAR enhanced the sensitivity to 5-FU-induced reduction of cellular viability and increased apoptosis in SGC-7901 cells. Furthermore, AICAR increased tumor suppressor genes [F-box and WD repeat domain containing 7 (FBXW7), semaphorin III/F (SEMA3F), and p21(Cip1) (p21)] but reduced mdr1 expression. Finally, p-AMPK levels were reduced in 5-FU-resistant gastric cancer cells compared to human immortalized gastric epithelial cell line and 5-FU-sensitive gastric cancer cells. AICAR not only induces apoptosis alone but also enhances pro-apoptotic effect of 5-FU in SGC-7901 cells, which lays an experimental foundation to develop AICAR as a chemotherapeutic sensitizer against gastric cancer.