Multiple myeloma (MM) cells express TLR. It has been shown that TLR ligands induce the proliferation, survival, and immune surveillance escape of MM cells through MyD88-TLR pathways. Deciphering TLR function in MM cells will help in understanding the mechanisms of tumor cell growth. In this study, we examined the response of MM cells to the MyD88-independent/TIR-domain-containing adapter-inducing IFN-beta-dependent TLR3. Deregulation of NF-kappaB pathway is a feature of MM cells, and we wondered whether TLR3 activation could mobilize the NF-kappaB pathway. We show that five of seven human myeloma cell line (HMCL) cells expressed TLR3. In the presence of the synthetic TLR3 ligand (poly(I:C)), activation of NF-kappaB pathway was observed in three of five selected TLR3(+) HMCL, NCI-H929, RPMI 8226, and KMM1. In agreement with NF-kappaB activation, only these three HMCL responded to poly(I:C), although by either an increase (KMM1) or a decrease (NCI-H929, RPMI 8226) of proliferation. We show that KMM1 increase of proliferation was prevented by NF-kappaB inhibitor. In contrast, inhibition of proliferation in both NCI-H929 and RPMI 8226 was due to IFN-alpha-induced apoptosis. We next demonstrated that p38 MAPK pathway controlled both IFN-alpha secretion and IFN-alpha-mediated cell death. Moreover, cell death also involved activation of ERK1/2 pathway. In conclusion, our results show that TLR3 ligand induces NF-kappaB pathway activation in MM and support a switching function of type I IFN in the functional outcome of TLR3 triggering in tumor cells.