Background Impairment of thiol/disulfide homeostasis, as well as vitamin D deficiency, are responsible for the pathophysiology of many acute and chronic diseases. This study examined the relationship between thiol/disulfide homeostasis and vitamin D level and supplementation. Methods A total of 203 healthy children were included in the study. The participants were divided into four groups according to 25-hydroxyvitamin D (25(OH)D) level: group 1, severe deficiency (mL); group 2, deficiency (10-20 ng/mL); group 3, insufficiency (20-30 ng/mL); and group 4, sufficiency (>= 30 ng/mL). Furthermore, group 5 was defined as being on vitamin D supplementation. Result Native thiol was lower in group 5 than in groups 2-4 (P = 0.003). Disulfide was higher in groups 1, 4 and 5 than groups 2 and 3 (P < 0.001). Total thiol was lower in group 5 than in group 4 (P = 0.032). The ratio of native thiol/total thiol was lower in groups 1 and 5 compared with groups 2 and 3, and in group 4 compared with group 3 (P < 0.001). The ratios of disulfide/total thiol and disulfide/native thiol were higher in groups 1 and 5 than in groups 2 and 3 whereas only the disulfide/total thiol ratio was higher in group 4 than in group 3 (P < 0.001). Conclusions In healthy children, severe deficiency of vitamin D causes impairment of thiol/disulfide homeostasis and increases protein oxidation, which cannot be reversed by external vitamin D supplementation.