Connexins and gap junctions play a crucial role during carcinogenesis. While diverse regulatory systems have been shown to modulate their function, the influence of cell motility on the intensity of gap junctional intercellular coupling has yet to be systematically addressed. Since cancer cell motility and intercellular coupling determine cancer development, we aimed at elucidating how mutual cell translocation modulates the intensity of gap junctional coupling in cell populations. Time-lapse analyses of the motility of connexin43 (Cx43)-coupled rat prostate carcinoma (MAT-LyLu) and mouse melanoma (B16) cells cultured on hyper-adhesive substrata revealed a reduced intensity of intercellular translocations in the two cell populations compared to the control conditions. While no detectable effects on the architecture of the actin cytoskeleton and Cx43 expression and phosphorylation were observed, the inhibition of cell motility was paralleled by an increase in the abundance of Cx43-positive plaques in cell-to-cell contacts and an enhancement of gap junctional coupling in cell populations cultured on hyper-adhesive substrata. Thus, a direct correlation between two cellular parameters crucial for carcinogenesis, i.e. cell motility and gap junctional coupling intensity exists in cancer cell populations.