Background: Dry eye is a disease (DED) of the tear film and ocular surface, leading to ocular discomforts and pain. The pathophysiology of DED includes tear film instability and ocular surface inflammation. Vitamin D refers to a group of fat-soluble secosteroids responsible for enhancing intestinal absorption of calcium, iron, magnesium, phosphate, and zinc. It is not strictly a vitamin, and may be considered a hormone as its synthesis and activity occur in different locations.Objective: This study aimed to investigate tear film function in patients with vitamin D deficiency. Patient and methods: A case-control study included 75 eyes of 39 patients who were referred to the Ophthalmologic Clinic, Menoufia University during the period from March 2020 to December 2020. Patients of dry eye underwent tear break up time, Schirmer I test without anesthesia and special tests as well as measurement of serum vitamin D level were assessed. Results: 25-hydroxy vitamin D level of the studied group ranged from 6-18.50 ng/ml with mean value of 8.22 ± 4.814 ng/ml. Also, majority of studied sample (92.3%) had bilateral affected eyes, followed by Gelinya (30%). Schirmer's test, tear film breakup time (TFBUT) and ocular surface disease index (OSDI) were increased significantly when compared the values at baseline and after treatment to be after 10 weeks (P < 0.001). Conclusion:Vitamin D replacement appears to improve ocular surface health in patients with vitamin D deficiency. However, further clinical trials with a large sample size and control group are warranted to define the role of vitamin D. Vitamin D supplementation is an effective and useful treatment for patients with dry eye syndrome (DES) that is refractory to conventional treatment.