Arsenite has been well-proved to act as both an environmental carcinogen as well as a tumor therapeutic agent. AP-1 is one of the transcription factors that can be induced upon arsenite stimulation. However, the study on the mechanism and the function of the arsenite-induced AP-1 transactivation remains far complete. Here we demonstrated that high dose of arsenite induced apoptotic response in mouse fibroblasts correlating with AP-1 transactivation, which events were mediated by both IKKalpha and IKKbeta, two major protein kinases responsible for NF-kappaB activation. In addition, the regulatory effect of IKKs on the arsenite-induced AP-1 activation was delivered by sequential induction of GADD45alpha expression and the activation of MAPKK (MKK3/4/6) and MAPK (JNK and p38K)-dependent pathways. We further provided evidence that p50, but not p65 subunit of NF-kappaB, was involved in GADD45alpha induction and the subsequent MAPKK/MAPK/AP-1 activation under arsenite exposure, while functional NF-kappaB induced by arsenite stimulation consisted of p65 but not of p50 subunit. Therefore, we concluded that both IKKalpha and IKKbeta can mediate arsenite-induced AP-1 transactivation through NF-kappaB activity-independent manner.