Introduction: Breast cancer accounts for 25% of all female cancer cases in India. Although, lipids and free radicals like Nitric Oxide (NO) have been related to breast cancer, but its pathogenesis remains controversial. Aim: To estimate NO, endothelial NO Synthase (eNOS) and lipid profile in serum of breast cancer patients and also to determine their possible role in patients of breast cancer in Indian women. Material and Methods: This was a hospital-based case-control study conducted for a period of 24 months from July 2012 to June 2014 in Dr VM Government Medical College, Solapur, with a total of 100 women (50 breast cancer cases and 50 healthy age matched controls) of age group 35-65 years. Fasting venous blood samples were collected. Biochemical parameters analysed in the study were serum NO, serum eNOS, serum Total Cholesterol (TC), serum Triglycerides (TG), serum High Density Lipoprotein (HDL) and serum Low Density Lipoprotein (LDL). Results were expressed as mean±Standard Deviation (SD) and Student’s unpaired t-test was used to compare the pairs of means. Results: There was no difference in mean age between cases (49.73±8.16 years) and controls (48.23±8 years). Serum levels of NO, eNOS, TC, TG and LDL were significantly higher in cases than in controls (55.92±6.54 µmol/L v/s 49.58±6.74 µmol/L for NO; 175.4±22.92 U/mL v/s 148.6±9.77 U/mL for eNOS; 271±9.16 mg/dL v/s 177.58±6.41 mg/dL for TC; 180.70±8.48 mg/ dL v/s 107.57±8.98 mg/dL for TG ; 177.39±6.21 mg/dL v/s 98.16±8.90 mg/dL for LDL). However, serum HDL levels did not showed significant difference in case group (57.55±6.40 mg/dL) when compared with control group (57.15±5.35 mg/dL). Conclusion: The present study demonstrated that high serum levels of NO, eNOS and lipids are associated with breast cancer and thereby suggests that increased NO, eNOS and abnormal lipid profile may have a role in pathogenesis of breast cancer.