Gliomas are particularly difficult to cure owing largely to their invasive nature. The neoplastic changes of astrocytes which give rise to these tumors frequently include a reduction of connexin43 (Cx43), the most abundant connexin isoform expressed in astrocytes. Cx43 is a subunit of gap junctions (GJ), intercellular channels which directly link the cytosol of adjacent cells and allow the regulated passage of ions and small molecules. To examine the role of Cx43 in glioma motility, we identified two variant C6 cell lines which endogenously express high (C6-H) or low (C6-L) levels of Cx43. In wound healing and transwell assays, C6-H cells were more motile than C6-L cells. To deduce whether Cx43 mediated these differences, assays were conducted on C6-H cells retrovirally transduced with Cx43 shRNA. Coincident with the stable knockdown of endogenous Cx43, a decrease in motility and invasion was observed. Gap junctional intercellular communication was also decreased, however motility assays conducted in the presence of GJ inhibitors did not reveal significant differences in cell motility. C6 cells transfected with full length or C-terminal truncated Cx43 (Cx43DeltaCT) were subjected to the aforementioned motility assays to expose alternate mechanisms of Cx43-mediated motility. Cells expressing full length Cx43 exhibited increased motility while cells expressing Cx43DeltaCT did not. This report, the first in which RNAi has been employed to reduce Cx43 expression in gliomas, indicates that the downregulation of Cx43 decreases motility of C6 cells. Furthermore, it is the first report to suggest that the Cx43 CT plays an important role in glioma motility.