We tested the correlation between the tightness of tight junctions (TJs) and the anti-invasive activity of the ethyl alcohol extract of Hizikia fusiforme (EHF) in Hep3B human hepatocarcinoma cells. EHF inhibited cell proliferation, motility, and invasiveness, which were associated with increased TJ tightness, as demonstrated by an increase in transepithelial electrical resistance. EHF dose-dependently decreased the secretion of matrix metalloprotease-2 and -9, which correlated with a decrease in mRNA and protein expression, but increased tissue inhibitor of metalloproteinase-1 and -2 mRNA levels. Additionally, immunoblotting results indicated that EHF suppressed the major components of TJ, claudins (-1, -3, and -4), which play a key role in the control and selectivity of paracellular transport. These data indicate that EHF may inhibit cancer cell invasion through the tightening of TJs, which may counteract the up-regulation of claudins. Furthermore, EHF treatment decreased the expression of insulin-like growth factor-1 receptor proteins, while concurrently increasing that of thrombospondin-1 and E-cadherin. In conclusion, these results suggest that EHF treatment may inhibit tumor metastasis and invasion and therefore act as a dietary resource for decreasing the risk of developing cancer.