Amadoriases I and II are deglycation isoenzymes from Aspergillus sp. of potential relevance for treatment of diabetic complications resulting from excessive protein glycation. Amadoriase II has a preference for anionic substrate with a Km of 0.23 and 2.53 mM for fructosylglycine and fructosylpropylamine, respectively. In contrast, the corresponding Km values for amadoriase I are 9.75 and 0.023 mM, respectively. Chemical modification of amadoriase II with p-hydroxyphenylglyoxal, a specific arginine-modifying reagent, resulted in an inhibition of enzyme activity toward fructosylglycine, while having less effect on the enzymatic activity toward fructosylpropylamine. Peptide mapping and subsequent mass spectrometry analysis suggest that Arg112 is one of the sites of p-hydroxyphenylglyoxal modification. Sequence alignment between amadoriase I and amadoriase II revealed that two glutamic acids in amadoriase I align to Arg112 and Arg114 in amadoriase II. Site-directed mutation of amadoriase II (R112E, R114E) resulted in reversal of the enzymatic activities toward fructosylglycine and fructosylpropylamine. Our results suggested that Arg112 and Arg114 are responsible for the high affinity of amadoriase II toward anionic substrates and determine the substrate selectivity of the enzyme.