To understand the mechanisms of photo-induced vitreous liquefaction, this study investigated the effects of free radicals on collagen and hyaluronic acid (HA). Bovine vitreous collagen or HA was irradiated by visible light in the presence of riboflavin (RF) as a photosensitizer. The changes in the molecular weight of collagen and HA were monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography, respectively. Free radicals were shown to cause an increase in the high-molecular-weight components and insolubilization of the vitreous collagen and a decrease in the molecular weight of HA. The change in molecular properties of the vitreous collagen could be attributed to extensive crosslinks of the molecules. Since RF is present in the vitreous, which is irradiated by visible light over a lifetime, both cross-links of vitreous collagen and degradation of HA may contribute to age-related vitreous liquefaction.