Purpose <p>To investigate the effects of vitamin D replacement therapy on dry eye disease (DED) test parameters in pediatric patients with vitamin D deficiency (VDD).</p> Study design <p>Prospective clinical study</p> Methods <p>This prospective study included 46 pediatric patients diagnosed with VDD and 39 sex- and age-matched healthy subjects. DED test parameters, including Schirmer test, tear break-up time (TBUT), upper and lower lid meiboscore and meibomian gland loss (MGL), tear meniscus height (TMH), tear meniscus area (TMA), Ocular Surface Disease Index (OSDI), and corneal fluorescein staining score (CFSS) were assessed. The measurements in the VDD group were performed twice, the first time before the treatment and the second time three months after the vitamin D replacement therapy. Only one assessment was made in the control subjects.</p> Results <p>Significant improvements were observed in the Schirmer test (p=0.007), TBUT (p=0.03), TMH (p&lt;0.001), TMA (p&lt;0.001), CFSS (p&lt;0.001), and OSDI scores (p=0.016) after vitamin D replacement therapy in children with VDD. Lower eyelid meiboscore and MGL improved significantly after treatment (p=0.022 and p&lt;0.001, respectively), but no significant difference was observed in upper meiboscore and MGL after vitamin D replacement therapy (p=0.986 and p=0.758, respectively). There was no significant difference between the treated VDD group and the healthy controls in Schirmer test, TBUT, TMH, TMA, OSDI scores and lower eyelid meiboscore and MGL (p&gt;0.005 for all).</p> Conclusion <p>Vitamin D replacement therapy significantly improves DED test parameters in pediatric patients with VDD.</p>