The β-isoform of protein kinase C (PKC) has paradoxically been suggested to be important for both insulin action and insulin resistance as well as for contributing to the pathogenesis of diabetic complications. Presently, we evaluated the effects of knockout of the PKCβ gene on overall glucose homeostasis and insulin regulation of glucose transport. To evaluate subtle differences in glucose homeostasis in vivo, knockout mice were extensively backcrossed in C57BL/6 mice to diminish genetic differences other than the absence of the PKCβ gene. PKCβ^−/− knockout offspring obtained through this backcrossing had 10% lower blood glucose levels than those observed in PKCβ^+/+ wild-type offspring in both the fasting state and 30 min after ip injection of glucose despite having similar or slightly lower serum insulin levels. Also, compared with commercially obtained C57BL/6–129/SV hybrid control mice, serum glucose levels were similar, and serum insulin levels were similar or slightly lower, in C57BL/6–129/SV hybrid PKCβ knockout mice in fasting and fed states and after ip glucose administration. In keeping with a tendency for slightly lower serum glucose and/or insulin levels in PKCβ knockout mice, insulin-stimulated 2-deoxyglucose (2-DOG) uptake was enhanced by 50–100% in isolated adipocytes; basal and insulin-stimulated epitope-tagged GLUT4 translocations in adipocytes were increased by 41% and 27%, respectively; and basal 2-DOG uptake was mildly increased by 20–25% in soleus muscles incubated in vitro. The reason for increased 2-DOG uptake and/or GLUT4 translocation in these tissues was uncertain, as there were no significant alterations in phosphatidylinositol 3-kinase activity or activation or in levels of GLUT1 or GLUT4 glucose transporters or other PKC isoforms. On the other hand, increases in 2-DOG uptake may have been partly caused by the loss of PKCβ1, rather than PKCβ2, as transient expression of PKCβ1 selectively inhibited insulin-stimulated translocation of epitope-tagged GLUT4 in adipocytes prepared from PKCβ knockout mice. Our findings suggest that 1) PKCβ is not required for insulin-stimulated glucose transport; 2) overall glucose homeostasis in vivo is mildly enhanced by knockout of the PKCβ gene; 3) glucose transport is increased in some tissues in PKCβ knockout mice; and 4) increased glucose transport may be partly due to loss of PKCβ1, which negatively modulates insulin-stimulated GLUT4 translocation.