Human mixed-lineage leukemia (MLL) family methyltransferases methylate histone H3 lysine 4 to different methylation states (me1/me2/me3) with distinct functional outputs, but the mechanism underlying the different product specificities of MLL proteins remains unclear. Here, we develop methodologies to quantitatively measure the methylation rate difference between mono-, di-, and tri-methylation steps and demonstrate that MLL proteins possess distinct product specificities in the context of the minimum MLL-RBBP5-ASH2L complex. Comparative structural analyses of MLL complexes by X-ray crystal structures, fluorine-19 nuclear magnetic resonance, and molecular dynamics simulations reveal that the dynamics of two conserved tyrosine residues at the "F/Y (phenylalanine/tyrosine) switch" positions fine-tune the product specificity. The variation in the intramolecular interaction between SET-N and SET-C affects the F/Y switch dynamics, thus determining the product specificities of MLL proteins. These results indicate a modified F/Y switch rule applicable for most SET domain methyltransferases and implicate the functional divergence of MLL proteins. [Display omitted] • MLL family methyltransferases possess distinct product specificities • Dynamics of the "F/Y switch" residues fine-tune the product specificity • Sequence variation in SET-N and SET-C interface affects the "F/Y switch" dynamics • The modified "F/Y switch" rule is applicable for most SET domain methyltransferases Li et al. establish a general criterion to define product specificity by comparing methylation rates of each step, enabling decoding the multifaceted product specificity of MLL family methyltransferases. They propose a modified "F/Y switch" rule applicable for most SET domain methyltransferases and provide a deeper understanding of dynamic histone methylation. [ABSTRACT FROM AUTHOR]