Objectives: Homocysteine (Hcy) is an endogenous amino acid containing sulfur, a product of the metabolism of the methionine essential amino acid. The increased risk of cardiovascular and malignant diseases is associated with elevated levels of total serum homocysteine (tHcy). The aim of this study was to evaluate serum homocysteine levels in patients with cholelithiasis and to look at the association between gallstone presence and total homocysteine level in North Eastern Uttar Pradesh. Methods: We included 100 patients of cholelithiasis who underwent cholecystectomy. Besides standard laboratory parameters, all the patients were measured for levels of triglyceride, high and low density lipoprotein cholesterol level, and total homocysteine. Results: Plasma homocysteine levels in all patients were elevated and found to be associated significantly (p<0.001) with Gallstone Disease (GSD) patients. Triglyceride and HDL showed also a significant statistical difference (p<0.001). In all patients with gallstone disease, total cholesterol and LDL concentrations were less than 240 mg / dl and 160 mg / dl, respectively. Conclusion: Plasma total homocysteine level was increased in gallstone disease patients. The risk of developing gallstone disease was associated with elevated total homocysteine levels.