The SET (SU(VAR)3-9, E(Z), and TRX) domain-containing MLL-family proteins (including MLL1/2/3/4 and SET1A/SET1B) specifically methylate H3K4 to play pivotal roles in transcriptional regulation of genes involved in hematopoiesis and development. The histone lysine methyltransferase (HKMT) activity of MLL1 is up-regulated by WDR5, RbBP5 and Ash2L, but the regulation mechanism remains elusive. Here we discover RbBP5-Ash2L heterodimer is the minimum unit to interact and activate all MLL proteins. The crystal structures of the active MLL3-RbBP5-Ash2L complex and MLL1-RbBP5-Ash2L reveal a two-step mechanism of MLL activation: a rigid-body rotation of SET-I (SET-Insertion) motif in MLL SET domain towards the substrate-binding groove upon RbBP5-Ash2L binding, followed by a substrate-induced local rearrangement in the SET-I motif to enclose the target lysine access channel for catalysis. The surprising identification of similar activation segment in both RbBP5 and many SET domains suggests a universal mechanism for histone methyltransferase regulation. Additionally, we characterized the distinct product specificities among different MLL proteins. These findings provide structural insights into the common theme and functional plasticity in complex assembly and activity regulation of MLL-family methyltransferases.