Hepatocyte growth factor (HGF) may impact the metastasis of prostate cancer via its action on prostate stem cells or their progeny. Tight junctions (TJs) are crucial to the process of metastasis and have been previously shown to be regulated by HGF. The present study aimed to evaluate the effect of HGF on the function of TJs in human prostate epithelial, prostate stem cell-like and prostate cancer cell lines. Four human prostate cancer cell lines (PC-3, DU-145, PZHPV-7, CaHPV-10), normal adult prostate parental epithelial cells (RWPE-1) and a stem cell-like derivative of RWPE-1 (WPE-STEM) were used to assess HGF-induced changes in TJs. A significant difference was noted in the behaviour between the WPE-STEM, RWPE-1 and the cancer cell lines which was HGF concentration-dependent. However, in the WPE-STEM cells, the effect was biphasic, with the cells seemingly resistant to HGF-modulated TJ disruption. Closer examination revealed that HGF affected the redistribution of ZO-1, ZO-2 and ZO-3 away from the TJs of confluent cells with concurrent loss of claudin-1 and claudin-5, and western blot analysis revealed a loss in TJ protein expression of ZO-1 and ZO-2. We demonstrated for the first time that HGF regulates TJ function in human prostate cells. Moreover, this regulation was dependent on the tumourigenicity of the cells, with the most aggressive cells most susceptible and the stem cell-like cells least susceptible. These data offer an intriguing glimpse of how TJs affect the behaviour of prostate cancer cells and how HGF modulates the expression and function of the molecules maintaining TJ structure and function.