Bone-forming osteoblasts have been recently reported capable of expressing the critical co-stimulatory molecule CD40 upon exposure to bacterial infection, which supports the unappreciated role of osteoblasts in modulating bone inflammation. Recent studies highlight the anti-inflammatory potential of glycogen synthase kinase-3β (GSK-3β) inhibitors; however, their effect on osteoblasts remains largely unclear. In the present study, we showed that treatment with SB216763, a highly specific GSK-3β inhibitor, resulted in a dose-dependent decrease in the mRNA and protein expression of CD40, as well as production of pro-inflammatory cytokines IL-6, TNF-α and IL-1β, in the Porphyromonas gingivalis-lipopolysaccharide (LPS)-stimulated murine osteoblastic-like MC3T3-E1 cells. Furthermore, inhibition of GSK-3β remarkably represses the LPS-induced activation of the nuclear factor kappa B (NF-κB) signaling pathway by suppressing IκBα phosphorylation, NF-κBp65 nuclear translocation, and NF-κBp65 DNA binding activity. Closer investigation by immunoprecipitation assay revealed that β-catenin can physically interact with NF-κBp65. The negative regulation effect of GSK-3β inhibitor on CD40 expression is mediated through β-catenin, for siRNA of β-catenin attenuated the GSK-3β inhibitor-induced repression of NF-κB activation and, consequently, the expression of CD40 and production of pro-inflammatory cytokines in LPS-stimulated MC3T3-E1 cells. Thus our results elucidate the molecular mechanisms whereby GSK-3β inhibitor prevents the LPS-induced CD40 expression on osteoblasts and provide supportive evidence of the potential role of GSK-3β inhibitors in suppressing the immune function of osteoblasts in inflammatory bone diseases.