Glucocorticoids (GCs) are often prescribed in clinics but many adverse effects are also attributed to GCs. It is important to determine the role of GCs in the development of those adverse effects. Here, we investigated the impact of GCs on trivalent chromium (Cr) distribution in animals. Cr has been proposed to be important for proper insulin sensitivity, and deficits may lead to disruption of metabolism. For comparison, the effect of a high-fat diet on Cr modulation was also evaluated. C57BL/6JNarl mice were fed regular or high-fat diets for 12 weeks and further grouped for treatment with prednisolone or saline. Cr levels in tissues were determined 12 h after the treatments. Interestingly, prednisolone treatment led to significantly reduced Cr levels in fat tissue in mice fed regular diets; compared to the high-fat diet alone, prednisolone plus the high-fat diet led to a further reduction in Cr levels in the liver, muscle, and fat. Notably, a single dose of prednisolone was linked with elevated Cr levels in the thigh bones of mice fed by either regular or high-fat diets. In conclusion, this report has provided evidence that prednisolone in combination with a high-fat diet effects modulation of Cr levels in selected tissues.