Livin is one of the most important members of the inhibitor of apoptosis protein family. It is overexpressed in several types of tumors and may have prognostic significance. The present study investigated the biological role of Livin in the oncogenic behavior of gastric cancer cells, the expression of Livin in gastric cancer tissue and the relationship of its expression with various clinicopathological parameters and patient survival. Small interfering RNA blocked Livin gene expression in AGS and SNU638 human gastric cancer cell lines. The expression of Livin was investigated in gastric cancer tissues by RT-PCR, western blotting and immunohistochemistry. The associations with various clinicopathological parameters and survival were analyzed. Livin knockdown inhibited tumor cell migration, invasion and proliferation in AGS and SNU638 cells. Livin knockdown induced apoptosis by activating caspase-3, caspase-7 and PARP. Livin knockdown induced cell cycle arrest by a decrease in cyclin D1, cyclin-dependent kinase 4 and 6 and an increase in expression of p21 and p27. The ERK1/2 and JNK signaling pathways were inhibited by Livin knockdown. Livin expression was upregulated in gastric cancer tissues at the mRNA and protein levels. However, no significant correlation was found between Livin expression and various clinicopathological parameters including survival. In conclusion, Livin expression may be important in the alteration of invasive and oncogenic phenotypes of gastric cancer cells. The prognostic relevance of Livin remains unclear.