Members of tumour necrosis factor (TNF) family usually trigger both survival and apoptotic signals in various cell types. Heat shock proteins (HSPs) are conserved proteins implicated in protection of cells from stress stimuli. However, the mechanisms of HSPs in TNFalpha-induced signalling pathway have not been fully elucidated. We report here that HSP70 over-expression in human colon cancer cells can inhibit TNFalpha-induced NFkappaB activation but promote TNFalpha-induced activation of c-Jun N-terminal kinase (JNK) through interaction with TNF receptor (TNFR)-associated factor 2 (TRAF2). We provide evidence that HSP70 over-expression can sequester TRAF2 in detergent-soluble fractions possibly through interacting with TRAF2, leading to reduced recruitment of receptor-interacting protein (RIP1) and IkappaB alpha kinase (IKK) signalosome to the TNFR1-TRADD complex and inhibited NFkappaB activation after TNFalpha stimuli. In addition, we found that HSP70-TRAF2 interaction can promote TNFalpha-induced JNK activation. Therefore, our study suggests that HSP70 may differentially regulate TNFalpha-induced activation of NFkappaB and JNK through interaction with TRAF2, contributing to the pro-apoptotic roles of HSP70 in TNFalpha-induced apoptosis of human colon cancer cells.