BACKGROUND Reg4, a member of the Reg multigene family, is highly upregulated in many gastrointestinal cancers including gastric cancer (GC). The enhanced expression of Reg4 is associated with the resistance of GC to 5-fluorouracil (5-FU), while the underlying mechanism is not clear. The aim of the present study was to explore the resistant mechanism underlying 5-FU resistance. MATERIAL AND METHODS Reg4 expression was assessed by Western blot analysis for SGC-7901, BGC-823, AGS, MKN28, and MKN45. Synthetic short single strand RNA oligonucleotides and Flag-Reg4 plasmid were used to investigate the biological function of Reg4 in vitro. The cell viability assay was performed by MTT. Flow cytometry was carried out to measure the apoptosis caused by 5-FU. Reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) was used to examine the expression of 5-FU metabolism related enzymes. The effect of Reg4 on intracellular signaling was evaluated by Western blot. RESULTS Western blot analysis of 5 GC cells showed that Reg4 was low or null in SGC-7901 and BGC-823, while high in AGS, MKN28, and MKN45. Over-expression of flag-Reg4 in SGC-7901 led to an increase in cell viability and lower apoptosis with 5-FU treatment. In contrast, siRNA knockdown of Reg4 enhanced 5-FU induced apoptosis. However, over-expression or knockdown of Reg4 had no significant influence on the expression of 5-FU metabolic enzymes. Further investigation revealed that Reg4 could activate Erk1/2-Bim-caspase3 cascade. CONCLUSIONS Reg4 inhibited apoptosis through regulating MAPK/Erk/Bim signaling pathway and thereby enhanced the resistance of GC to 5-FU.