To compare the plasma levels of homocysteine between patients with keratoconus and healthy subjects. Thirty-three keratoconus patients, and 47 age-gender matched healthy subjects were included in this prospective study. The plasma level of homocysteine, folic acid, and vitamin B12 was assessed using the chemiluminescence immunometric method. According to the manufacturer's instructions, the normal plasma level of homocysteine, vitamin B12 and folic acid was accepted as ≤ 13 μm/L, 191-663 pg/mL, and 4.6-18.7 ng/mL respectively. Mann-Whitney U and Spearman's correlation tests were used for pairwise comparisons and correlation analysis, respectively. There was a statistically significant difference between keratoconus patients and healthy subjects in terms of mean plasma level of homocysteine (15.02 ± 8.01 μm/l in keratoconus patients versus 12.62 ± 8.17 μm/l in healthy subjects, p = 0.01). However, the mean plasma level of either vitamin B12 (263.78 ± 107.2 pg/ml in keratoconus patients versus 264.78 ± 94.2 pg/ml in healthy subjects, p = 0.3), or folic acid (5.98 ± 3.2 ng/ml in keratoconus patients versus 6.72 ± 3.1 ng/ml in healthy subjects, p = 0.1) were not statistically significantly different between two groups. A negative correlation was found between plasma homocysteine level and central corneal thickness (p < 0.001). A positive correlation was found between plasma homocysteine level and steepest keratometry (p = 0.004) and average Sim-K (p = 0.002). The increased plasma level of homocysteine in keratoconus patients may either arise from a consequence of biochemical events such as oxidative stress, or it may contribute to the pathogenesis or progression of keratoconus by chelating copper, which is an important co-factor of lysyl oxidase enzyme taking place in corneal collagen crosslinking.