Connexins assemble to form gap junction channels to mediate communication and signaling between adjacent cells by allowing the passage of ions, metabolites and signaling molecules. It has been demonstrated that activation of protein kinase C (PKC) can lead to phosphorylation of connexin 43 (Cx43), which affect trafficking, assembly, degradation, and gating of Cx43 gap junction channels. On the other hand, it has been shown that cholesterol plays important role in the proper function of Cx43. Therefore, we hypothesize that cholesterol can regulate the function of Cx43 through the PKC dependent pathway. In the present study, using methly-β-cyclodextrin (MβCD) to manipulate membrane cholesterol content, we studied effect of cholesterol on the function and distribution of Cx43 in H9c2 cell line, a standard cardiac cell line derived from embryonic cells. Immunofluorescent images showed that cholesterol enrichment made the Cx43 distribution from condensed to diffused manner in the interface between the cells. Cholesterol enrichment not only increased phosphorylated state of Cx43 (p-Cx43) that could be reduced by PKC antagonists, but also decreased dye transfer ability that could be reversed by treatment of PKC antagonists.Furthermore, cholesterol depletion increased dye transfer ability and decreased p-Cx43 level in the cells pre-treated with the PKC agonist. In addition, p-Cx43 level at Ser368 upregulated by cholesterol enrichment could be downregulated by PKC antagonists. These results suggested that cholesterol could enhance activity of PKC to phosphorylate Cx43 at Ser368, resulting in Cx43 gap junction disassembly and reduction of the cell-to-cell communication.