Mevalonate kinase plays a key role in regulating the biosynthesis of cholesterol in animal cells. Human mevalonate kinase His20Pro has been reported as one of the three common mutations in the mevalonate kinase gene in mevalonic aciduria and hyperimmunoglobulinemia D/periodic fever syndrome patients. His20 is also a highly conserved residue among all aligned mevalonate kinase sequences. To study the role of His20 of mevalonate kinase, a variety of mutant expression plasmids of rat mevalonate kinase including pRMK(H20L), pRMK(H20Y), and pRMK(H20K) were constructed using site-directed mutagenesis, and mutant proteins were overexpressed and purified. CD spectroscopy of wild-type protein and mutants indicated that mutations H20L and H20Y did not induce significant secondary structural changes. The results from kinetic studies showed that this highly conserved histidine is an important residue for the function of the enzyme.