The identification of proinflammatory signal transduction pathways may suggest new therapeutic targets. In this study, we examine which signaling pathways are involved in tumor necrosis factor (TNF)-induced matrix metalloproteinase 9 (MMP9) secretion in human chorionic trophoblast (CT) cells. Purified CT cells were cultured in the presence of antibodies or chemical inhibitors that specifically block/inhibit distinct TNF receptors and kinase pathways. TNF-induced proMMP9 production, as measured by zymography, was significantly blocked/inhibited by TNF receptor 1 (TNFRSF1A) antibody, NFKB activation inhibitor (NFKBAI), and MAPK1/3 (ERK) inhibitor (U0126) (P < 0.01), but not by TNF receptor 2 (TNFRSF1B) antibody, MAPK14 (p38 MAPK) inhibitor (SB203580), and MAPK8/9/10 (JNK) inhibitor (SP600125). By Western blot analysis, we found that TNF rapidly and significantly increased phosphorylation of IKBKB, MAPK1/3, and MAPK8/9/10 and that the phosphorylation of these kinases by TNF was reduced significantly by TNFRSF1A neutralizing antibody, but not by TNFRSF1B neutralizing antibody. Moreover, we found that TNF increased TNF receptor-associated factor (TRAF) 1 and decreased TRAF2 protein expression through TNFRSF1A, but not TNFRSF1B. The CT cells that had increased TRAF1 and decreased TRAF2 after an initial TNF treatment demonstrated a dramatic deficiency in phosphorylation of the above protein kinases following a secondary TNF treatment. Localization of RELA subunit by immunocytochemistry was shifted to the nuclei after TNF treatment compared to cytosol in untreated controls. We also found cross-talk between the phosphoinositide 3-kinase pathway and ERK pathway. In summary, we have demonstrated that TNF stimulates proMMP9 production in CT cells through TNFRSF1A-TRAFs-IKBKB-NFKB and ERK signaling pathways, but not through TNFRSF1B and JNK/p38-AP-1 pathways.