Purpose To evaluate conjunctival surface cells and tear-film functions in cases with vitamin D deficiency. Methods Thirty-six patients with serum vitamin D deficiency and 27 control subjects with normal serum vitamin D levels were included in this prospective study. The tear break-up time (TBUT), Schirmer II test, and conjunctival impression cytology tests were performed to all participants. Results The mean serum vitamin D levels were 10.5 +/- 5.0 mu l in the study group and 33.9 +/- 11.7 mu l in the control group (p < 0.001). The median TBUT scores were 11 s and 17 s and the median Schirmer II values were 7.5 mm and 12 mm in the study and the control groups, respectively (p = 0.003, p = 0.049). According to the Nelson staging system, 69.4% of the patients in the study group had grade 2 or 3 impression cytology, whereas 18.5% of the participants in the control group had grade 2 or 3 impression cytology (p < 0.001). Conclusion Our study results showed that vitamin D deficiency may lead to dry eye causing conjunctival squamous metaplasia and loss of goblet cells on the ocular surface.