Pseudoexfoliation (PEX) is a multifactorial age-related disease involving deposition of extracellular fibrillar material on anterior ocular tissues. If left untreated, the early stage of deposition termed as pseudoexfoliation syndrome (PEXS) may lead to the advanced stage of pseudoexfoliation glaucoma (PEXG) characterised by increased intraocular pressure, damage to the optic nerve and subsequent irreversible blindness. The etiology of PEX is complex and identification of novel factors associated with the disease is needed. This study aimed to identify the involvement of vimentin in pseudoexfoliation pathology and assess the levels of vimentin, clusterin and fibulin-5 in the circulating fluids of PEX patients compared to controls. Eighty-seven participants (35 controls, 35 PEXS and 17 PEXG) were enrolled for this case-control study. The expression of vimentin in lens capsules of patients and age-sex matched controls was assayed by qRT-PCR, western blotting and immunohistochemistry. Aqueous humor (AH) vimentin levels and plasma levels of vimentin, clusterin and fibulin-5 were assayed through ELISA. Increased vimentin was observed in the lens capsule of patients compared to controls at mRNA and protein levels. Compared to control (11.5 ± 1.4 ng/ml [±SEM]), the AH vimentin concentrations were significantly higher (ANOVA p = 0.01) in PEXS (16.4 ± 1.8 ng/ml) and PEXG (20.1 ± 2.5 ng/ml). Compared to controls (372.2 ± 15.1 ng/ml), plasma vimentin levels were significantly higher (ANOVA, p < 0.001) in PEXS (449.9 ± 15.7 ng/ml) and PEXG (535.5 ± 25.0 ng/ml). The plasma and aqueous humor levels of vimentin showed a positive correlation of 0.31. The plasma levels of clusterin were 298.9 ± 19.0 μg/ml, 367.8 ± 25.6 μg/ml and 272.9 ± 16.8 μg/ml in controls, PEXS and PEXG, respectively and were significantly higher in PEXS (p = 0.03) compared to control. Plasma fibulin-5 levels were 149 ± 32.2 pg/ml, 187.6 ± 32.3 pg/ml and 203.8 ± 27.3 pg/ml in controls, PEXS and PEXG, respectively and there was no significant difference in its levels between the groups (ANOVA p = 0.49). In conclusion, vimentin is upregulated in PEX affected eyes. Increased vimentin levels in plasma and AH differentiate PEXS and PEXG from controls.