Hemochromatosis factor E (HFE) is a member of class I MHC family and plays a significant role in the iron homeostasis. Denaturation of HFE induced by guanidinium chloride (GdmCl) was measured by monitoring changes in []222 (mean residue ellipticity at 222 nm), intrinsic fluorescence emission intensity at 346 nm (F346) and the difference absorption coefficient at 287 nm287) at pH 8.0 and 25 °C. Coincidence of denaturation curves of these optical properties suggests that GdmCl-induced denaturation (native (N) state ↔ denatured (D) state) is a two-state process. The GdmCl-induced denaturation was found reversible in the entire concentration range of the denaturant. All denaturation curves were analyzed for 〖∆G〗_D^0, Gibbs free energy change associated with the denaturation equilibrium (N state ↔ D state) in the absence of GdmCl, which is a measure of HFE stability. We further performed molecular dynamics simulation for 40 ns to see the effect of GdmCl on the structural stability of HFE. A well defined correlation was established between in vitro and in silico studies.