A proliferation-inducing ligand (APRIL) is a key factor involved in the tumor development and progression in some tumor tissues and cells. Its overexpression and as gene target in SW480 colon carcinoma cells was confirmed in our previous study. To seek a more potent way to treat colon carcinoma using a gene therapy method, herein, we constructed a multiple short hairpin RNA (shRNA) expression vector containing four shRNAs against the APRIL gene in SW480 cells. APRIL expression levels and cell biological behavior were detected after transfection with different kinds of vectors. As expected, we found that our multiple shRNA vector produced a more significant knockdown effect of APRIL than the vectors containing only one APRIL shRNA. Furthermore, our findings indicate that silencing APRIL expression in SW480 cells decreased their malignancy by reducing proliferation, invasion and adhesion, as well as inducing apoptosis. Based on our findings, vectors containing multiple shRNAs to silence the expression of APRIL may be exploited as a novel therapeutic strategy for tumors.