Protein kinase C (PKC) plays an essential role in cellular signal transduction for mediating a variety of biological functions. There are 11 PKC isoforms and these isoforms are believed to play distinct roles in cells. Although the role of individual isoforms of PKC has been investigated in many fields, little is known about the role of PKC in osteoblastic differentiation. Here, we investigated which isoforms of PKC are involved in osteoblastic differentiation of the mouse preosteoblastic cell line MC3T3-E1. Treatment with Gö6976, an inhibitor of PKCα and PKCβI, increased alkaline phosphatase (ALP) activity as well as gene expression of ALP and Osteocalcin (OCN), and enhanced calcification of the extracellular matrix. Concurrently, osteoblastic cell proliferation decreased at a concentration of 1.0 μM. In contrast, a PKCβ inhibitor, which inhibits PKCβI and PKCβII, did not significantly affect osteoblastic differentiation or cell proliferation. Knockdown of PKCα using MC3T3-E1 cells transfected with siRNA also induced an increase in ALP activity and in gene expression of ALP and OCN. In contrast, overexpression of wild-type PKCα decreased ALP activity and attenuated osteoblastic differentiation markers including ALP and OCN, but promoted cell proliferation. Taken together, our results indicate that PKCα suppresses osteoblastic differentiation, but promotes osteoblastic cell proliferation. These results imply that PKCα may have a pivotal role in cell signaling that modulates the differentiation and proliferation of osteoblasts.