Abstract: Osteosarcoma is the most common primary tumor of bone, occur mainly during childhood and adolescence. No reports are available in literature where serum folate and vitamin B12 have been assessed in osteosarcoma patients. Hence, the present study was planned to analyze status of folate and vitamin B12 in thirty osteosarcoma patients and compare it with thirty controls (subjects with musculoskeletal pain). Serum calcium, alkaline phosphatase levels were higher and phosphorus levels were comparable in osteosarcoma patients as compared to controls. Serum folate levels were significantly raised in osteosarcoma patients as compared to controls (p<0.001). Serum vitamin B12 levels were significantly lowered in osteosarcoma patients as compared to controls (p<0.001). Folate-mediated one-carbon metabolism (FOCM) is unequivocally linked to multiple health outcomes, including birth defects, several types of cancer and possibly cardiovascular disease and cognitive function. Tumors are known to up regulate FRα modulating the folate uptake in serum. Vitamin B12 diminishes osteoblastic activity and lowered serum vitamin B12 levels are possibly due to increased metabolic demand of the tumor. The present study suggests that these parameters can serve as useful markers for diagnosis and follow up of disease.