Hypothermia provides an effective neuro and cardio-protection in clinical settings implying ischemia/reperfusion injury (I/R). At the onset of reperfusion, succinate-induced reactive oxygen species (ROS) production, impaired oxidative phosphorylation (OXPHOS), and decreased Ca2+ retention capacity (CRC) concur to mitochondrial damages. We explored the effects of temperature from 6 to 37 °C on OXPHOS, ROS production, and CRC, using isolated mitochondria from mouse brain and heart. Oxygen consumption and ROS production was gradually inhibited when cooling from 37 to 6 °C in brain mitochondria (BM) and heart mitochondria (HM). The decrease in ROS production was gradual in BM but steeper between 31 and 20 °C in HM. In respiring mitochondria, the gradual activation of complex II, in addition of complex I, dramatically enhanced ROS production at all temperatures without modifying respiration, likely because of ubiquinone over-reduction. Finally, CRC values were linearly increased by cooling in both BM and HM. In BM, the Ca2+ uptake rate by the mitochondrial calcium uniporter (MCU) decreased by 2.7-fold between 25 and 37 °C, but decreased by 5.7-fold between 25 and 37 °C in HM. In conclusion, mild cold (25–37 °C) exerts differential inhibitory effects by preventing ROS production, by reverse electron transfer (RET) in BM, and by reducing MCU-mediated Ca2+ uptake rate in BM and HM.