It has been shown that cholesterol modulates activity of protein kinase C (PKC), and PKC phosphorylates connexin 43 (Cx43) to regulate its function, respectively. However, it is not known whether cholesterol modulates function of Cx43 through regulating activity of PKC. In the present study, we demonstrated that cholesterol enrichment reduced the dye transfer ability of Cx43 in cultured H9c2 cells. Western blot analysis indicated that cholesterol enrichment enhanced the phosphorylated state of Cx43. Immunofluorescent images showed that cholesterol enrichment made the Cx43 distribution from condensed to diffused manner in the interface between the cells. In cholesterol enriched cells, PKC antagonists partially restored the dye transfer ability among the cells, downregulated the phosphorylation of Cx43 and redistributed Cx43 from the diffused manner to the condensed manner in the cell interface. In addition, reduction of cholesterol level suppressed PKC activity to phosphorylate Cx43 and restored Cx43 function in PKC agonist-treated cells. Furthermore, we demonstrated that cholesterol enrichment upregulated the phosphorylated state of Cx43 at Ser368, while PKC antagonists reversed the effect. Taken together, cholesterol level in the cells plays important roles in regulating Cx43 function through activation of the PKC signaling pathway.