18Beta-glycyrrhetinic acid (18beta-GA) regulates serine/threonine dephosphorylation of connexin43 (Cx43). Phospho-specific antibodies were used here to determine the effect of 18beta-GA on serine 368-phosphorylated Cx43 (pSer368Cx43) in cultured rat neonatal cardiomyocytes by immunofluorescence microscopy and immunoblot analyses. 18beta-GA caused a time-dependent increase in pSer368Cx43 levels and induced gap junction disassembly, shown by a change in pSer368Cx43 immunostaining from large aggregates to dispersed punctates at cell-cell contact areas. 18beta-GA also induced a time-dependent increase in the levels of serine 729-phosphorylated PKCepsilon, the active form of PKCepsilon. The 18beta-GA-induced increase in pSer368Cx43 levels and changes in pSer368Cx43 staining pattern were abolished by the PKC inhibitor, chelerythrine. Furthermore, 18beta-GA increased the co-immunoprecipitation of Cx43 with PKCepsilon. However, the 18beta-GA-induced increase in pSer368Cx43 levels and increased association of Cx43 with PKCepsilon were inhibited by co-treatment with the protein phosphatase type 1 and type 2A inhibitor, calyculin A. We conclude that 18beta-GA induces Ser368 phosphorylation of Cx43 via PKCepsilon.