Vitamin D3 and calcium deficiency are influential factors in the development of osteoporosis. Also, osteocalcin is considered to be one of the markers for osteoporosis as it is produced primarily by osteoblasts during bone formation. The aim of this study is to assess the serum levels of vitamin D3, osteocalcin, and calcium with bone mineral density to differentiate between osteopenic and osteoporotic postmenopausal women. A case control study that included 120 postmenopausal Iraqi women divided into three groups [40 postmenopausal women with osteoporosis, 40 postmenopausal women with osteopenia, and 40 post-menopausal women free from osteoporosis and osteopenia as control group. The results obtained showed that vitamin D3 was significantly lower in both osteoporosis and osteopenia groups when compared to the control group. The average levels of osteocalcin were significantly lower in osteoporosis and osteopenia groups when compared to the control. Also, the average levels of serum calcium were significantly lower in osteoporosis and osteopenia groups when compared to the control group. The results of ROC curve analysis demonstrated excellent ability of the included tests variables with AUC= 0.912, 1.00, 0.91 and 1.00 for osteocalcin, vitamin D3, calcium, and T-score, respectively that can classify osteoporosis from control groups, and the cut-off values of these parameters could be used for this differentiation. Also, the results demonstrated excellent ability of all the included tests with the AUC = 0.902, 1.00, 0.912, 1.00 for osteocalcin, vitamin D3, calcium, and T-score, respectively that can classify osteopenia from control groups with the cut-off values. While, the results showed that only T-score (AUC= 1.00) is the best parameter used to differentiate osteoporosis from osteopenia. In conclusion, levels of vitamin D3, osteocalcin and calcium could be used to give an indication of bone mineral density status in osteopenic or osteoporotic postmenopausal women without the use of a DEXA analysis. But, the differentiation between osteopenia and osteoporosis was only obtained by the DEXA analysis method.