Osteoporosis is a chronic disease of public health importance, particularly in low and middle income countries. Measuring the bone mineral balance (BMB) in a non-invasive manner, and its response to different interventions, is critical to the definition of optimal strategies for its prevention and management. In this study, we demonstrate the usefulness of natural variability in calcium isotopes (δ44/40Ca) of urine and the derived BMB estimates as a biomarker of bone health and its responsiveness to interventions. Vitamin D3 is commonly used as a supplement for the prevention and treatment of osteoporosis, along with calcium supplements. We studied the effect of a short term vitamin D3 supplementation on changes in urine δ44/40Ca and the derived BMB. δ44/40Ca before and after the vitamin D3 supplementation yielded a statistically significant change (p = 0.050) with a positive δ44/40Ca enrichment. The mean derived BMB was net positive (0.04 ± 0.05) in comparison to a net negative value for the control group (-0.03 ± 0.01). These results indicate the potential usefulness of urinary natural δ44/40Ca and the derived BMB, which, along with bone mineral density could be used as a sensitive marker for precision in the prevention and treatment of osteoporosis.