Canonical Wnt signaling is critical for melanocyte lineage commitment and melanoma development. RAD6B, an ubiquitin-conjugating enzyme critical for translesion DNA synthesis (TLS) potentiates β-catenin stability/activity by inducing proteasome-insensitive polyubiquitination. RAD6B expression is induced by β-catenin, triggering a positive feedback loop between the two proteins. RAD6B function in melanoma development/progression was investigated by targeting RAD6B using CrispR/Cas9 or a RAD6-selective small molecule inhibitor SMI#9. SMI#9 treatment inhibited melanoma cell proliferation but not normal melanocytes. RAD6B knockout or inhibition in metastatic melanoma cells downregulated β-catenin, β-catenin-regulated MITF, SOX10, and vimentin proteins, and MITF-regulated Melan A. RAD6B knockout or inhibition decreased migration/invasion, tumor growth and lung metastasis. RNA-seq and stem cell pathway real-time RT-PCR analysis revealed profound reductions in WNT1 expressions in RAD6B knockout M14 cells compared to control. Expression of β-catenin-regulated genes VIM, MITF-M, Melan A and TYRP1 (a tyrosinase family member critical for melanin biosynthesis) were reduced in RAD6B knockout cells. Pathway analysis identified gene networks regulating stem cell pluripotency, Wnt signaling, melanocyte development, pigmentation signaling and protein ubiquitination besides DNA damage response signaling were impacted by RAD6B gene disruption. These data reveal an important and early role for RAD6B in melanoma development besides its bonafide TLS function, and suggest that targeting RAD6B could potentially provide a novel strategy to treat melanomas with dysregulated canonical Wnt signaling.Copyright © 2020. Published by Elsevier Inc.