Skeletal homeostasis is crucial to maintaining bone quality. In the absence of balanced bone formation and bone resorption, skeletal disease develops. Numerous studies show that the gap junction protein connexin43 (Cx43) plays a role in the function of bone forming osteoblasts and osteocytes, as well as the coordination of bone resorption by these cells. Little is known about how Cx43 influence skeletal homeostasis. In particular, the identities of the second messenger signals that are communicated by bone cells to regulate skeletal homeostasis are unclear. Here, we assessed the role of cAMP as a biologically relevant second messenger communicated by Cx43 containing gap junctions among bone cells and its regulation of genes involved in skeletal homeostasis, including sclerostin and RANKL in UMR106- cells. Methods: UMR106-cells were cultured and transfected with different plasmid constructs to manipulate cAMP levels (constitutively active GalphaS), as well as Cx43 expression. Luciferase reporter assays and western blots were used to assess cAMP-dependent signaling. Regulation of the SOST and RANKL were assessed by quantitative RT-PCR. Results: The in vitro studies conducted have shown the synergism between Cx43 and G-alphaS, which generates cAMP, confirming the amplified signal when these two were expressed together. Further, a modified parachute assay revealed that cell-to-cell contact was required for the sharing of cAMP-dependent signals by Cx43 expressing bone cells. Cx43 and cAMP combine to potently increase RANKL (a stimulator of bone resorption) expression and suppress SOST expression (An inhibitor of bone formation). Conclusion: There was up regulated signals when Cx43 and G-alphaS were co-expressed implying the role of the second messenger cAMP in cell-to-cell communication by bone cells. In addition, these signals converged on the expression of RANKL and sclerostin, two genes that play a major role in bone turnover. Accordingly, this pathway may represent one of the ways that gap junctions regulate skeletal homeostasis and could be a target of therapeutic intervention for skeletal disease.