αA-Crystallin is a small heat shock protein that functions as a molecular chaperone and a lens structural protein. The R49C single-point mutation in αA-crystallin causes hereditary human cataracts. We have previously investigated the in vivo properties of this mutant in a gene knock-in mouse model. Remarkably, homozygous mice carrying the αA-R49C mutant exhibit nearly complete lens opacity concurrent with small lenses and small eyes. Here we have investigated the 90° light scattering, viscosity, refractive index, and bis-ANS fluorescence of lens proteins isolated from the αA-R49C mouse lenses and found that the concentration of total water-soluble proteins showed a pronounced decrease in αA-R49C homozygous lenses. Light scattering measurements on proteins separated by gel permeation chromatography showed a small amount of high-molecular mass aggregated material in the void volume which still remains soluble in αA-R49C homozygous lens homogenates. An increased level of binding of β- and γ-crystallin to the α-crystallin fraction was observed in αA-R49C heterozygous and homozygous lenses but not in wild-type lenses. Quantitative analysis with the hydrophobic fluorescence probe bis-ANS showed a pronounced increase in fluorescence yield upon binding to α-crystallin from mutant as compared with the wild-type lenses. These results suggest that the decrease in the solubility of the αA-R49C mutant protein was due to an increase in its hydrophobicity and supra-aggregation of αA-crystallin that leads to cataract formation. Our study further shows that analysis of mutant proteins from the mouse model is an effective way to understand the mechanism of protein insolubilization in hereditary cataracts.