The total concentration of calcium in bile is much higher (25 meq/lt) than the plasma concentration due to calcium binding by other components of bile, especially bile salts. Vitamin D plays an important role in regulation of bile salts and megalin thus preventing gallstones. Since an increase in Biliary Calcium was found during gallstone formation in various studies. In present study an attempt was done to see if Vitamin D deficiency causes a rise in Biliary Calcium and precipitating gallstones.In present study a negative poor correlation was also observed between Biliary Calcium and Serum Calcium (r= -0.02) i.e. if Serum Calcium levels fall the Biliary Calcium level may increase but such finding was not significant statistically (p=0.83). In all patients calcium in bile was higher than serum suggesting the ability of gallbladder to concentrate calcium in gallbladder bile that may lead to stone formation.In the present study the mean Serum Vitamin D3 level was 13.16±6.76 ng/ml which shows deficiency of vitamin D3 in patients of gallstones.