Background: Vitamin D deficiency is an unrecognized epidemic and a common health problem worldwide. Studies carried in Saudi Arabia showed a high prevalence in pediatrics, but there was no published data in Northern Border area. Aim: The aim of this study was to determine the prevalence of vitamin D deficiency among female students in our university, Kingdom of Saudi Arabia (KSA). Methods: This study was a cross-sectional survey. The study was conducted at our university in KSA from January 2015 to September 2015. We selected a sample of 247 students to participate after taking their consent. We used A Questionnaire to determine the demographic characteristics, awareness of vitamin D sources, exposure to sunlight and dietary habits. We calculated the body mass index, collected blood samples of 5 ml, stored them at minus 20-degree centigrade and analyzed them. We measured Serum concentration of 25-hydroxycholecalciferol 25(OH)D using enzyme-linked immuno-sorbent assay (ELISA). Serum calcium, phosphorous and parathyroid hormone were also measured. We analyzed the data using Statistical Package for Social Sciences software produced by The International Business Machines (IBM-SPSS) Version 22. The P value of < 0.05 was considered statistically significant. Results: The mean age of the study group was 22.2996 (2.68) years. The mean 25-hydroxycholecalciferol level was 7.59 (SD 1.9) ng/ml. All the participants (100%) were found to have vitamin D deficiency defined as 25(OH)D ≤ 20 ng/ml. Severe vitamin D deficiency was found in 220 (89.1%). Most of the participants were aware of the sources of vitamin D 88.7% and most of them take food containing calcium and phosphorus but only 23 participants (9.3%) were exposed to sunlight adequately. Conclusion: Vitamin D deficiency was highly prevalent among the study group.