In order to maximize the efficiency and versatility of the vector-based siRNA approach, we have developed a novel siRNA expression vector containing multiple tandem siRNA cassettes to investigate the   synergistic inhibitory effect of it on human colon cancer cells proliferation.  Multiple siRNA recombinant    expression vector targeting simultaneously Livin and Survivin genes was constructed and transfected into human colon cancer cell.  The effect of multiple siRNA recombinant expression vector was detected by RT-PCR,  Western blotting and flow cytometry.  It was confirmed by restriction endonuclease and sequence analysis   that multiple siRNA recombinant expression vector targeting simultaneously Livin and Survivin genes was   constructed successfully.  Livin and Survivin genes inhibition ratio of Livin and Survivin siRNA at mRNA levels were 27.90% and 32.24%, at protein levels were 22.28% and 40.86%, the apoptotic ratio was (11.69 ± 1.37) %, but the synergistic effect was weaker than Livin and Survivin RNA interference, respectively.  The multiple siRNA recombinant expression vector targeting simultaneously Livin and Survivin genes has been constructed successfully.  It can inhibit the expression of Livin and Survivin genes in human colon cancer cells, but the  synergistic effect was weaker than Livin and Survivin RNA interferences, respectively.