Tight junctions in epithelial cells act as cell—cell adhesion structures and govern paracellular permeability. Disruption of these functions can lead to dissociation of cancer cells. This study aimed to determine whether HGF, a cytokine secreted by stromal cells, is capable of modulating expression and function of tight junction molecules in human breast cancer cell lines. HGF decreased trans-epithelial resistance and increased paracellular permeability of two human breast cancer cell lines, MDA MB 231 and MCF-7. Q-PCR showed that HGF modulated the levels of several tight junction molecule (occludin, claudin-1 and −5, JAM−1 and −2) mRNA transcripts in MDA MB 231 and MCF-7 cells. Western blotting and immunohistochemistry also showed modulation of expression of the tight junction molecule, occludin. It is suggested that HGF disrupts tight junction function in human breast cancer cells by effecting changes in the expression of tight junction molecules at both the mRNA and protein levels. We conclude that regulation of tight junctions could be of fundamental importance in the prevention of metastasis of breast cancer cells.