Livin, a novel member of the human inhibitors of apoptosis protein family, has been shown to be critical for tumor progression and poor prognosis for several types of malignancies. However, limited reports exist regarding the biological functions of Livin in human gastric cancer (GC). The present study investigated the clinical significance of Livin and caspase-3 (CAS-3) in human GC using immunohistochemistry assay, and explore the potential using RNA interference to knockdown Livin expression, including the subsequent effects on tumor growth and invasion in GC cells in vitro and in vivo. Our results showed that the rate of positive expression of Livin was significantly higher in GC tissues compared to that in adjacent non-cancer tissues (ANCT) (64.1 vs. 30.8%, P<0.001), while CAS-3 was lower in GC tissues than in ANCT (33.3 vs. 66.7%, P=0.001). Livin expression was positively correlated with tumor differentiation and lymph node metastases (P=0.009; P=0.007), while CAS-3 was negatively correlated with them (P=0.036; P=0.002) in patients with GC. Furthermore, knockdown of Livin inhibited cell proliferative activities and invasive potential, and induced cell in situ apoptosis in GC cells, accompanied with decreased expression of p38 MAPK, VEGF and MMP-2 and increased expression of CAS-3. In addition, the tumor volumes in the SGC7901 subcutaneous nude mouse model treated with Lv-shLivin was significantly smaller compared to those of the PBS group (P<0.01). Taken together, our findings indicate that the expression of Livin is increased in human GC and correlates with tumor differentiation and lymph node metastases, while knockdown of Livin inhibits cell growth and invasion through blockade of the MAPK pathway in GC cells, suggesting that Livin may be a potential therapeutic target for the treatment of GC.