Functional adipocyte glucose disposal is a key component of global glucose homeostasis. PKC beta II is involved in rat skeletal muscle cell ISGT. Western blot analysis and real-time PCR revealed 3T3-L1 cells developmentally regulated PKC beta splicing such that PKC beta I was downregulated and PKC beta II was upregulated during the course of differentiation. An initial glucose uptake screen using PKC inhibitor LY379196 pointed to a PKC isozyme other than PKC zeta mediating 3T3-L1 adipocyte ISGT. Subsequent use of PKC beta II inhibitor CGP53353 pointed to a role for PKC beta II in ISGT. Western blot analysis showed that CGP53353 specifically inhibited phosphorylation of PKC beta II Serine 660. Subcellular fractionation and immunofluorescence demonstrated that PKC beta II regulates GLUT4 translocation. Further Western blot, immunofluorescence and co-immunoprecipitation analysis reveal that PKC beta II inhibition does not affect mTORC2 activity yet abrogates phosphorylation of Akt Serine 473. PKC beta II regulates GLUT4 translocation by regulating Akt phosphorylation and thus activity. Published by Elsevier Inc.