Chemoresistance is a major obstacle to cancer therapy including that of colon cancer (CC). Although the dysregulation of many miRNAs has been implicated in 5-fluorouracil (5-FU) resistance in CC cells, the specific role of miR-20b in chemoresistance has not been documented. In the present study, we first determined the expression of miR-20b by RT-PCR and the levels of a disintegrin and metalloprotease 9 (ADAM9) and epidermal growth factor receptor (EGFR) by western blotting in CC and adjacent non-cancerous tissues from 5-FU-sensitive or -resistant CC patients. Subsequently, 5-FU-sensitive (HCT116) and -resistant (HCT116-R) cells were obtained, and the levels of miR-20b, ADAM9 and EGFR were detected. Meanwhile, the 5-FU resistance of the cells was examined by assessing cell viability (by MTT assay) and apoptosis (by flow cytometry). After transfection of miR-20b into HCT116-R cells, drug resistance was reexamined. We then confirmed the relationship between miR-20b and ADAM9 by luciferase reporter assay. Finally, 5-FU resistance in HCT116 and HCT116-R cells was compared after transfection with miR-20b. Our results showed that miR-20b was expressed at lower levels in the 5-FU-resistant tissues and cells than in the 5-FU-sensitive tissues and cells. The opposite was the case for expression of ADAM9 and EGFR. In addition, we demonstrated that ADAM9 is a direct target of miR-20b and that miR-20b decreased the 5-FU resistance of HCT116-R cells. Our findings suggest that miR-20b reduces 5-FU resistance to induce apoptosis in vitro by suppressing ADAM9/EGFR in CC cells.