Regulation of connexin43 (Cx43) expression affects cell proliferation, differentiation and apoptosis in a gap junctional intercellular communication (GJIC)-independent manner. However, the underlying mechanisms of Cx43-mediated cell cycle suppression are still poorly understood. To elucidate the molecular mechanism of Cx43-mediated cell cycle suppression, we searched for Cx43 interacting proteins by using a proteomics approach. Here, we have identified a Cx43-interacting protein, heat shock cognate protein 70 (Hsc70). We confirmed that Hsc70 directly binds to the C-terminus of Cx43, whereas Hsc54, a splice variant of Hsc70, does not, that Cx43 competes with cyclin D1 for binding to Hsc70, and that the nuclear accumulation of cyclin D1 is reduced by overexpression of Cx43 in a GJIC-independent manner, which is restored by co-overexpression with Hsc70. As a result, the cell proliferation is regulated by Cx43. Our results suggest that Cx43-Hsc70 interaction probably plays a critical role during G1/S progression.