Vitamin D is a fat-soluble steroid hormone. It obtained from foods or by the synthesis in the skin after sunlight exposure (Ultraviolet rays) and converted to the active form in the liver and kidneys. Vitamin D3 is highly affected by hormones of the menstrual cycle.  Some evidence also suggests a possible role for vitamin D in the regulation of sexual function. There is no scientific evidence present that shows a relationship between marital status and vitamin D3 levels. The aim of this study is to investigate the correlation between vitamin D3 levels and marital status in females of different age groups in a given population. Methodology: A total of 140 females were randomly selected from the outpatient department in Al-Sader teaching hospital in Al-Najaf province Iraq in the period of December 2018 and March 2019. Venous blood (5 ml) was drawn from all subjects.  Subjects were classified according to their marital status (single, married) and age groups. Subjects taking vitamin D3 supplementation were discarded from the study.  Vitamin D3 and calcium analysis were determined from Human serum using ELISA microtiter plate. Calcium levels mg/dl were also determined using serum (Sysmex XP-300 Atomic Hematology Analyzer). Results: The results of this study showed that all females of all age groups suffered from hypovitaminosis D3 due to bone disorders. The levels of vitamin D3 in all age groups single females were significantly (P<0.05) lower than that in married subjects. Serum calcium levels were statistically significant (P<0.05) lower in females that are single compared to married females. Conclusion: Females that were single had lower vitamin D3 levels as well as lower serum calcium levels compared to those married. The outcome of this study shows that married females have higher vitamin D3 levels compared to that of single subjects.