Insulin produced by islet β-cells plays a critical role in glucose homeostasis, with type 1 and type 2 diabetes both resulting from inactivation and/or loss of this cell population. Islet-enriched transcription factors regulate β-cell formation and function, yet little is known about the molecules recruited to mediate control. An unbiased in-cell biochemical and mass spectrometry strategy was used to isolate MafA transcription factor–binding proteins. Among the many coregulators identified were all of the subunits of the mixed-lineage leukemia 3 (Mll3) and 4 (Mll4) complexes, with histone 3 lysine 4 methyltransferases strongly associated with gene activation. MafA was bound to the ∼1.5 MDa Mll3 and Mll4 complexes in size-fractionated β-cell extracts. Likewise, closely related human MAFB, which is important to β-cell formation and coproduced with MAFA in adult human islet β-cells, bound MLL3 and MLL4 complexes. Knockdown of NCOA6, a core subunit of these methyltransferases, reduced expression of a subset of MAFA and MAFB target genes in mouse and human β-cell lines. In contrast, a broader effect on MafA/MafB gene activation was observed in mice lacking NCoA6 in islet β-cells. We propose that MLL3 and MLL4 are broadly required for controlling MAFA and MAFB transactivation during development and postnatally.