The aim of current study is the risk assessment of biochemical variables of medical importance, vitamin D and calcium, in pathogenesis of Diabetes Mellitus Type II (T2DM) human beings and the risk assessment due to variation of these biochemical variables in diabetic patients. One hundred subjects (n=100) were recruited from different hospitals in Lahore (31.5204° N, 74.3587° E) after taken informed consents individually. This study was approved by the ethical committee of Institute of Molecular Biology and Biotechnology (IMBB), The University of Lahore, Lahore. Subjects were equally divide in two groups; diabetic (case group; n=50) and non-diabetic (control; n=50). Calcium and vitamin D levels were measured by the biochemical laboratory of Hospitals. Finally the data was statistically analyzed by using SPSS software (Version 16). A total of 52 % males and 48 % females were investigated for changes in serum calcium and vitamin D in relation to their biochemical importance in Diabetes Mellitus. Both calcium and vitamin D levels were significantly low in diabetic group (p=0.003 and p=0.001 respectively) as compare to control group. Persons correlation showed calcium and vitamin D were directly proportional to each other (R2= 0.577). Odds ratio showed that the low vitamin D and low Calcium levels were 3.47 and 3.17 time more prevalent in diabetic group as compare to the non- diabetic group. Diabetes Mellitus is an increasing health issue in world causing severe morbidity and mortality. So it is important to investigate the non-conventional risk factors of Diabetes Mellitus. Thus, the current study illuminates the risk assessment of altered levels of vitamin D and calcium, their possible pathogenesis in Diabetes Mellitus. It can be concluded from this study that supplementation of vitamin D and calcium till to the normal physiological limits can help not only lowering the risk of diabetes but may also prevent its progression.