Diffuse large B-cell lymphoma (DLBCL), the most common lymphoma, shows either no response or development of resistance to further treatment in 30% of the patients that warrants the development of novel drugs. We have reported that ON 01910.Na (rigosertib), a multikinase inhibitor, is selectively cytotoxic for DLBCL and induces more hyperphosphorylation and sumoylation of Ran GTPase-activating protein 1 (RanGAP1) in DLBCL cells than in non-neoplastic lymphoblastoid cell line. However, the exact mechanism of rigosertib-induced cell death in DLBCL remains to be clarified. Here, we analyzed the efficacy of rigosertib against DLBCL cells in vitro and in vivo and its molecular effects on tumor biology. We found for the first time that rigosertib attenuated expression of unmodified and sumoylated tumor necrosis factor receptor–associated factor 6 (TRAF6) and c-Myb and inhibited nuclear entry of sumoylated RanGAP1, TRAF6, and c-Myb that was confirmed by immunofluorescence. Moreover, co-immunoprecipitation showed that rigosertib induced sequestration of c-Myb and TRAF6 in the cytoplasm by stimulating their sumoylation through the RanGAP1*SUMO1/Ubc9 pathway. Specific knockdown of c-Myb and TRAF6 induced tumor cell apoptosis and cell cycle arrest at G1 phase. Xenograft mice bearing lymphoma cells also exhibited effective tumor regression on rigosertib treatment along with cytoplasmic expression of c-Myb and TRAF6. Nuclear expression of c-Myb in clinical cases of DLBCL correlated with a poor prognosis. Thus, suppression of c-Myb and TRAF6 activity may have therapeutic implication in DLBCL. These data support the clinical development of rigosertib in DLBCL.