Jumonji domain containing iron (II), 2-oxoglutarate (2OG)-dependent dioxygenases from Jmjd2 family demethylate trimethylated histone3-lysine 9 (H3-K9me3), and also H3-K9me2 and H3-K36me3, albeit at lower rates. Recently, we have identified the first non-histone substrates of JmjD2 demethylases. Here, we studied the substrate specificity of Jmjd2a-c demethylases using site-directed mutagenesis and novel non-histone substrates. We identified preference of Arg at -1 position and a smaller amino acid at -2 position using both singly and doubly mutated peptide substrates by Jmjd2a-c demethylases. Our results also identified similarities in substrate selectivity by H3-K9 methyltransferase, G9a and Jmjd2 demethylases despite their distinct reaction mechanisms. Published by Elsevier Inc.