Cordycepin, a specific polyadenylation inhibitor, is the main functional component in Cordyceps militaris that is one of the top three renowned traditional Chinese medicines. In this study, we performed in vitro experiments to investigate the anti-invasive and anti-metastatic activities of cordycepin using human prostate carcinoma LNCaP cells. Cordycepin were administered and their effects on LPS-induced cell migration and invasion by wound healing migration assay, measurement of TER and In vitro invasiveness assay. Within the concentrations which were not cytotoxic effects, cordycepin caused a concentration-dependent suppression of LPS-induced cell migration and invasion. The anti-invasive activity of cordycepin was also found to be associated with increased tightness of the TJ, which was confirmed by an increase in TER. The activity of MMP-2 in LNCaP cells was dose-dependently inhibited by treatment with cordycepin, and this was also correlated with a decrease in expression of its mRNA and proteins, and up-regulation of TIMPs expression. Additionally, cordycepin repressed the LPS-induced NF-kB activation and phosphorylation of PI3K/AKT. Taken together, these findings suggest that cordycepin inhibited LPS-induced migration and invasion of LNCaP cells by down-regulating the expression and activity of MMP-2, and the possible targets may be NF-kB and PI3K/AKT.