The stable calcium (Ca) isotopes offer a minimally invasive method for assessing Ca balance in the body, providing a new avenue for research and clinical applications. In this study, we measured the Ca isotopic composition of soft tissues (brain, muscle, liver, and kidney), mineralized tissue (bone), and blood (plasma) from 10 mice (5 females and 5 males) with three different genetic backgrounds and same age (3 months old). The results reveal a distinctive Ca isotopic composition in different body compartments of mice, primally controlled by each compartment’s unique Ca metabolism and genetic background, independent of sex. The bones are enriched in the lighter Ca isotopes (δ⁴⁴/40Ca_bone =  − 0.10 ± 0.55 ‰) compared to blood and other soft tissues, reflecting the preferential incorporation of lighter Ca isotopes through bone formation, while heavier Ca isotopes remain preferentially in blood. The brain and muscle are enriched in lighter Ca isotopes (δ⁴⁴/40Ca_brain =  − 0.10 ± 0.53 ‰; δ⁴⁴/40Ca_muscle = 0.19 ± 0.41 ‰) relative to blood and other soft tissues, making the brain the isotopically lightest soft tissues of the mouse body. In contrast, the kidney is enriched in heavier isotopes (δ⁴⁴/40Ca_kidney = 0.86 ± 0.31 ‰) reflecting filtration and reabsorption by the kidney. This study provides important insight into the Ca isotopic composition of various body compartments and fluids. Graphical Abstract