The aim of this study was to elucidate the localization of RhoA in human cancer tissues and cancer cell lines. Immunohistochemistry and immunofluorescence microscopy were used to determine the localization of RhoA in cancer tissues and cell lines, respectively. Western blotting was used to determine the quantity of RhoA in cell nucleus, cytosol and membrane. Immunofluorescence microscopy revealed that in all cell lines examined in this experiment (including SGC-7901, Hela, A549 and SW480), RhoA was localized not only on the membrane, in the cytosol, but also in the nucleus. Within the nucleus its precise localization was in the nucleolus. In the transformed gastric epithelial cell line GES, RhoA was also found to be localized on the membrane, in the cytosol and the nucleus, but the nuclear location was less than that of cancer cell lines. Immunohistochemistry microscopy revealed that in cancer tissues the nuclear RhoA was obviously more than that of para-cancer tissue; in the cancer tissues undergoing inflammation and necrosis, nuclear RhoA was even higher. RhoA is generally distributed in the nucleus of cells and the distribution increases when the cells undergo tumorigenesis, suggesting a new role for RhoA.