Background: Among all vitamins, Vitamin D is important with respect to bone health and body immunity. Hypovitaminosis D is a global health issue, and considered as an epidemic. The worldwide, a billion of people with hypovitaminosis D has been estimated, however, there are a limited number of studies has been conducted in third world countries to measure the Vitamin D levels in their general population. This investigative study aimed to determine the prevalence of hypovitaminosis D in general population of District D. G. Khan, Pakistan. Methodology: After a personal visit in various hospitals, 100 patients with vitamin D deficiency and insufficiency were enrolled in the study. They full filling inclusion and exclusion criteria. After taking informed written consent, blood samples were drawn and examine the serum levels of 25-hydroxy vitamin D. The serum level of 25-hydroxy vitamin D below 32 ng/ml is classified as Vitamin D insufficiency, while below 20 ng/ml is Vitamin D deficiency. Results: The mean age of 62 male (18-50) years and 38 females (18-50) years. Among these, serum 25(OH) Vitamin D means level of 62 male samples while 38 female samples were studied. The 27 males showed vitamin insufficiency, 25 males were Vitamin D deficient. Among 38 females, 11 subjects showed vitamin D insufficiency, 22 were vitamin D deficient, while 10 males and 5 females were normal control. The male subject showed significantly low serum calcium and phosphorous level, but elevated Alkaline Phosphatase (ALP) level as compared to normal male control. Similar finding has been found in female subjects. Conclusion: The vitamin D deficiency or insufficiency reduces the serum calcium and phosphorous level, while it causes elevation of serum ALP level.