A significant association between increased level of blood homocysteine (hyperhomocysteinimia) and various eye pathological disorders including cataract has been reported. This metabolic byproduct is converted into a highly reactive cyclic thioester compound, homocysteine thiolactone (HCTL), which can potentially react with free amino groups in protein. In the current study, as bovine lens γ-Crystallin (γ-Cry) was incubated with HCTL, various spectroscopic techniques, gel mobility shift assay, and microscopic analysis were applied to characterize structural variation and aggregation of this protein. According to the fluorescence results, HCTL-induced structural alteration was accompanied with the significant enhancement in surface hydrophobicity of γ-Cry. Also, this cyclic thioester was indicated to alter γ-Cry secondary structures and to induce aggregation of this protein. The results of gel mobility shift assay suggest the involvement of disulfide bond cross-linking in formation of the protein aggregates. In conjunction with Thioflavin T and Congo red assays, the microscopic analysis also suggests that HCTL can induce formation of ordered aggregate entities in bovine lens γ-Cry. The relationship between γ-Cry insolubilization/aggregation and growth of cataract disorders has been already reported. Therefore, the induction of structural alteration and aggregation of γ-Cry by HCTL can elucidate the pathomechanism underlying cataract disorders particularly in hyperhomocysteinimia.