Although the connexin32 (Cx32)-mediated gap junction is abolished in hepatocellular carcinoma (HCC), the expression of cytoplasmic Cx32 tends to increase in correspondence with the grade of malignancy. Establishing a Tet-off expression system in human nonmetastatic HuH7 HCC cells where cytoplasmic Cx32 was overexpressed by doxycycline (Dox) withdrawal, we previously demonstrated that overexpression of cytoplasmic Cx32 made HuH7 cells metastatic in mice. In our study, hypothesizing that the cytoplasmic Cx32-induced metastasis may involve expansion of the cancer stem cell (CSC) population, we examined whether cytoplasmic Cx32 controlled the size of the side population (SP) in HuH7 Tet-off Cx32 cells. Fluorescence-activated cell sorting revealed that SP was expanded in a Dox-free medium compared with a Dox-supplemented one. Although cytoplasmic Cx32 did not block maturation from SP to non-SP, purified SP reconstituted a larger SP fraction in the Dox-free medium than in the Dox-supplemented one. Furthermore, although SP from HuH7 Tet-off mock cells formed a similar number of CSC spheres of a similar size whether with or without Dox, SP from HuH7 Tet-off Cx32 cells developed a greater number of larger CSC spheres in the Dox-free medium than in the Dox-supplemented one. Taken together, these results suggest that accumulation of cytoplasmic Cx32 should enhance self-renewal of CSC to expand the CSC population in HCC.