Survivin has attracted abundant interest in tumor research since it was discovered in 1997. However, several studies indicate that the relationship between survivin expression and tumor behavior is still not fully understood. Among the current methods available, proteomics is an effective platform to globally detect and characterize proteins. Thus, we constructed the recombinant adenovirus [ad-survivin/ short hairpin RNA (shRNA)], which contains shRNA of survivin, and transfected it into SW480 cells. Then, we detected survivin gene expression after shRNA interference, and its influence on apoptosis and the cell cycle was analyzed. A comparative proteomic approach was used to identify the differential proteins between SW480/survivin (-) and SW480/survivin (+) cells. The results showed that survivin was expressed at a high level in SW480 cells and that the subcellular localization was observed in the cytoplasm. Recombinant adenovirus could suppress survivin-expression efficiency and induce apoptosis by affecting mitosis. The differentially expressed proteins identified by two-dimensional proteome analysis were related to various cellular programs involving cell proliferation, cell cycle, apoptosis, expression of nucleic acid metabolic genes, and the regulation of signal transduction. The proteomic approach implemented here offers a powerful tool for identifying novel tumor markers. Survivin plays an important role in controlling tumor growth by a variety of molecular regulatory mechanisms. Inhibition of survivin expression could effectively inhibit tumor growth.