The oncogenic transcription factor c-Maf has been proposed as an ideal therapeutic target for multiple myeloma (MM) but how to achieve it is still elusive. In the present study we found the Otub1/c-Maf axis could be a potential target. Otub1, an OTU family deubiquitinase, was found to interact with c-Maf by mass spectrometry. Otub1 abrogates c-Maf K48-linked polyubiquitination thus preventing its degradation and enhancing its transcriptional activity. Specifically, this deubiquitinating activity depends on its Lys71 and the N-terminus but independent UBE2O, a known E2 of c-Maf. Otub1 promotes MM cell survival and MM tumor growth. In contrast, silence of Otub1 leads to c-Maf degradation and c-Maf-expressing MM cell apoptosis. Therefore, the Otub1/c-Maf axis could be a therapeutic target of MM. In order to explore this concept, we performed a c-Maf-recognition element-driven luciferase-based screen against FDA-approved drugs and natural products, from which the generic cardiac glycoside lanatoside C (LanC) is found to prevent c-Maf de-ubiquitination and induces its degradation by disrupting the interaction of Otub1 and c-Maf. Consequently, LanC inhibits c-Maf transcriptional activity, induces c-Maf-expressing MM cell apoptosis, and suppresses MM growth and prolongs overall survival of model mice but without apparent toxicity. Therefore, the present study identifies Otub1 as a novel deubiquitinase of c-Maf and establishes that the Otub1/c-Maf axis is a potential therapeutic target for MM. Copyright © 2020 American Society of Hematology.