Dietary conjugated linoleic acid (CLA) has shown wide biologically beneficial effects, such as anticancer, antiatherosclerotic, antidiabetic, immunomodulating, and antiobesity effects. However, the effects of CLA on total body ash, reflective of bone mineral content, have not been consistent. We hypothesized that the inconsistency of the CLA effect on ash may be linked to interaction between CLA and dietary calcium levels. Thus, we investigated the effects of CLA on body ash in conjunction with various calcium levels. Male ICR mice were fed three different levels of calcium (0.01, 0.5, and 1%) with or without 0.5% CLA for 4 weeks for Experiment 1 and separate CLA isomers at 0.22% level with 1% calcium in Experiment 2. CLA feeding reduced body fat regardless of dietary calcium level, whereas CLA supplementation increased body ash compared to control only in animals fed the 1% calcium. In Experiment 2 it was confirmed that this observation was associated with the trans-10, cis-12 CLA isomer, but not with the cis-9, trans-11 isomer. CLA administration with 1% dietary calcium significantly improved total ash percent (%) in femurs, confirming that CLA has the potential to be used to improve bone mass.