The mechanism by which TPA-induced PKC activity modulates osteoclastogenesis is not clear. Using a RAW264.7 cell culture system and assays for NF-κB nuclear translocation, NF-κB reporter gene activity, and MAPK assays, we demonstrated that TPA inhibits osteoclastogenesis through the suppression of RANKL-induced NF-κβ activation. Introduction: The protein kinase C (PKC) pathway has been suggested to be an important regulator of osteoclastic bone resorption. The role of PKC in RANKL-induced osteoclastogenesis, however, is not clear. In this study, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, on osteoclastogenesis and studied its role in RANKL-induced signaling. Materials and Methods: RANKL-induced RAW264.7 cell differentiation into osteoclast-like cells was used to assess the effect of TPA on osteoclastogenesis. Assays for NF-κB nuclear translocation, NF-κB reporter gene activity, protein kinase activity, and Western blotting were used to examine the effects of TPA on RANKL-induced NF-κβ, c-Jun N-terminal kinase (JNK), and MEK/ERK and p38 signal transduction pathways. Results: We found that TPA inhibited RANKL-induced RAW264.7 cell differentiation into osteoclasts in a dose-dependent manner. Time course analysis showed that the inhibitory effect of TPA on RANKL-induced osteoclastogenesis occurs predominantly at an early stage of osteoclast differentiation. TPA alone had little effect on NF-κβ activation in RAW264.7 cells, but it suppresses the RANKL-induced NF-κβ activation in a dose-dependent fashion. Interestingly, the suppressive effect of TPA on RANKL-induced NF-κβ activation was prevented by a conventional PKC inhibitor, Go6976. Supershift studies revealed that the RANKL-induced DNA binding of NF-κβ complexes consisted of C-Rel, NF-κB1 (p50), and RelA (p65). In addition, TPA induced the activation of JNK in RAW264.7 cells but had little effect on RANKL-induced activation of JNK. TPA also inhibited RANKL-induced activation of ERK but had little effect on p38 activation. Conclusion: Given that NF-κB activation is obligatory for osteoclast differentiation, our studies imply that inhibition of osteoclastogenesis by TPA is, at least in part, caused by the suppression of RANKL-induced activation of NF-κβ during an early stage of osteoclastogenesis. Selective modulation of RANKL signaling pathways by PKC activators may have important therapeutic implications for the treatment of bone diseases associated with enhanced bone resorption.