Abstract: The antitumor activities of NL-101, aHDACi/DNA damage dual-targeting drug, on human multiple myeloma in vitro and in vivo were studied. Furthermore, the primary mechanisms were revealed. We detected the anti-proliferative activity of NL-101 on 10 human multiple myeloma cell lines, and the combinational effect of NL-101 and bortezomib on RPMI 8226 cell line. The inducing effects of NL-101 on cell cycle arrest and apoptosis were detected by FACS. The effects of NL-101 on acetyled-Histone H3, total Histone H3, acetyled α-Tubulin, total α-Tubulin, phospho-Histone H2A. X and total Histone H2A. X were evaluated by Western blott. We also demonstrated the antitumor activity of NL-101 and the combinational effect of NL-101 and bortezomib on RPMI 8226 xenograft tumor model in vivo. Results showed that NL-101 possessed strong antitumor activities on human multiple myeloma cells in vitro and in vivo. NL-101exhibited significant HDAC inhibitory activity and DNA alkylating activity. NL-101not only inhibited histone deacetylation level, but also increased the DNA damage in multiple myeloma cells. Meanwhile, NL-101 induced cell cycle arrest and apoptosis. Also, the synergistic effect of NL-101 was discovered when combined with bortezomib in vitro and in vivo. These data demonstrated that NL-101 may be a potent agent for the treatment of human multiple myeloma in future.