Gadolinium-based compounds are most widely utilized for paramagnetic contrast agents, but, the toxicological mechanism of gadolinium (Gd) had not been fully elucidated since the first report about Gd anomaly. In this work, we analyzed the effect of Gd3+ on mitochondria in vitro by microcalorimetry. Microcalorimetry can provide detailed kinetic and thermodynamic information from thermogenic curve. At the tested concentration, Gd3+ induced the increase of growth rate constant (k1). At high concentration (100–500 μM), the maximum power output time (tm), the decline rate constant (−k2) and the time of activity recovery phase (tR) decreased with the addition of Gd3+ and the maximum power output (Pm) increased. At low concentration (0–100 μM), the changes were different from high concentration. From the results we concluded that the effect of different concentrations of Gd3+ had a relationship with time, high concentration of Gd3+ induced mitochondrial energy metabolism disturb however low concentration may promote mitochondrial adaption to physiological stresses. The effect of low concentration of Gd3+ need more work to elucidate the mechanism. The results of total heat output (Q) and mitochondrial respiratory activities suggested high concentrations of Gd3+ could accelerate adenosine triphosphate (ATP) consumption under respiratory system damaged.