Osteoblasts are affected by TNF-α overproduction by immune cells during inflammation. It has been suggested that functional NF-κB sites are involved in TNF-α-induced bone resorption. Thus, we explored the effect of pyrrolidine dithiocarbamate (PDTC), which potently blocks the activation of nuclear factor (NF-κB), on the induction of TNF-α-induced activation of JNK/SAPK, AP-1, cytochrome c, caspase and apoptosis in MC3T3E1 osteoblasts. Pretreatment of the cells with PDTC blocked TNF-α-induced NF-κB activation. TNF-α-induced activation of AP-1, another nuclear transcription factor, was suppressed by PDTC. The activation of c-Jun N-terminal kinase, implicated in the regulation of AP-1, was also down regulated by PDTC. TNF-α-induced apoptosis, release of cytochrome c and subsequent activation of caspase-3 were abolished by PDTC. TNF-α-induced apoptosis was partially blocked by Ac-DEVD-CHO, a caspase-3 inhibitor, suggesting that caspase-3 is involved in TNF-α- mediated signaling through NF-κB in MC3T3E1 osteoblasts. Thus, these results demonstrate that PDTC, has an inhibitory effect on TNF-α-mediated activation of JNK/SAPK, AP-1, cytochrome c release and subsequent caspase-3, leading to the inhibition of apoptosis. Our study may contribute to the treatment of TNF-α-associated immune and inflammatory diseases such as rheumatoid arthritis and periodontal diseases.