We characterized the effects of a newly developed signal transducers and activators of transcription 3 (STAT3) inhibitor, LLL12 in multiple myeloma (MM) cells. LLL12 specifically inhibited STAT3 phosphorylation, nuclear localization, DNA binding activity, down-regulated STAT3 downstream genes, and induced apoptosis in MM cells. Importantly, LLL12 significantly inhibited STAT3 phosphorylation, induced apoptosis in primary MM cells which came from patients that were clinically resistant to lenalidomide and bortezomib. LLL12 is a potent inhibitor of cell proliferation with IC50 values ranging between 0.26 and 1.96 mu M in MM and primary MM cells. LLL12 also inhibited STAT3 phosphorylation induced by interleukin-6 (IL-6) and interferon-a but not STAT1, STAT2, STAT4 and STAT6 phosphorylation induced by interferon-a, interferon-? and IL-4 indicating the selectivity of LLL12 for STAT3. The selectively of LLL12 on STAT3 was further demonstrated on 21 protein kinases, which LLL12 had IC50 values =73.92 mu M. In addition, the pretreatment of LLL12 blocked the promotion of the cell proliferation and resistance to lenalidomide by IL-6. Furthermore, LLL12 significantly blocked tumor growth of MM cells in mouse model. Our results indicate that LLL12 blocks constitutive STAT3 and IL-6 induced STAT3 signaling and may be a potential therapeutic agent for MM.