Mitochondrial 2,4-dienoyl-CoA reductase is a key enzyme for the beta-oxidation of unsaturated fatty acids. Sequence alignment indicates that there are five highly conserved acidic residues, one of which might act as a proton donor. We constructed five mutant expression plasmids of human mitochondrial 2,4-dienoyl-CoA reductase using site-directed mutagenesis. Mutant proteins were overexpressed in Escherichia coli and purified with a nickel metal affinity column. Studies of these mutant proteins were carried out, and the proton donor is likely to be E276. Three substrate analogs were synthesized and characterized. Two analogs, 2-fluoro-2,4-octadienoyl-CoA and 5-methyl-2,4-hexadienoyl-CoA, were substrates of the enzyme. Another analog, 3-furan-2-yl-acrylyl-CoA, was not a substrate, but a competitive inhibitor of the enzyme. These studies increased our understanding of human mitochondrial 2,4-dienoyl-CoA reductase.