Myeloid differentiation primary response protein 88 (MYD88) is a critical universal adapter that transduces signaling from the Toll-like receptors and interleukin receptors to downstream NF-kB. MYD88L265P (leucine changed to proline at position 265) is a gain-of-function mutation occurred frequently in B-cell malignancies such as Waldenstrom macroglobulinemia. Here we show that an E3 ligase RING finger protein family 138 (RNF138) catalyzed K63-linked non-proteolytic polyubiquitination of MYD88L265P, resulting in enhanced recruitment of interleukin 1 receptor-associated kinases and elevated NF-kB activation. However, RNF138 had little effect on wild-type MYD88 (MYD88WT). With either RNF138 knockdown or mutation on MYD88 ubiquitination sites, MYD88L265P was unable to constitutively activate NF-kB. A20, a negative regulator of NF-kB signaling, mediated K48-linked polyubiquitination of RNF138 for proteasomal degradation. Depletion of A20 further augmented MYD88L265P-mediated NF-kB activation and lymphoma growth. Furthermore, A20 expression was negatively correlated with RNF138 expression and NF-kB activation in lymphomas with MYD88L265P and in those without. Strikingly, RNF138 expression was positively correlated with NF-kB activation in lymphomas with MYD88L265P, but not in those without MYD88L265P. Collectively, our study reveals a novel mutation-specific biochemical reaction that drive B-cell oncogenesis, providing a therapeutic opportunity for targeting oncogenic MYD88L265P, while sparing MYD88WT that is critical to innate immunity.