Ca2+ entry via the Mitochondrial Calcium Uniporter (MCU) participates in energetic adaption to workload under physiological conditions but is thought to contribute to cell death during ischemia-reperfusion (I/R) injury. We have previously shown that mitochondrial membrane potential (ΔΨm) instability contributes to early-reperfusion arrhythmias and contractile dysfunction; however, the role of mitochondrial Ca2+ (mCa2+) uptake in triggering ΔΨm oscillation is unclear. Here, by acutely knocking out MCU, we examine whether MCU-mediated mCa2+ uptake is required to trigger ΔΨm loss or oscillation during early reperfusion in neonatal mouse ventricular myocyte (NMVM) monolayers. We monitored mCa2+ (with MitoCam) and ΔΨm (with TMRM) in WT(MCUfl/fl) and MCU-KO (MCUfl/fl+AdCre) NMVMs during in vitro I/R (previously described by Solhjoo et al JMCC 2015) by confocal microscopy. Image sequences were analyzed for changes in mCa2+ and ΔΨm by segmenting individual cells in ImageJ. To quantify ΔΨm oscillations in mitochondrial clusters during reperfusion, a new wavelet-transform-based analysis was developed using MATLAB's wavelet toolbox. Surprisingly, our findings demonstrate that MCU knockout does not significantly alter mCa2+ import during I/R, nor does it affect ΔΨm recovery during Reperfusion. In fact, MCU-KO moderately shortened the latency to Ischemic ΔΨm depolarization. In contrast, blocking the mitochondrial sodium-calcium exchanger (mNCLX) with CGP-37157 suppressed the mCa2+ increase during Ischemia. Moreover, blocking mNCLX also did not affect ΔΨm recovery during Reperfusion or the frequency of ∆Ψm oscillations, confirming that mitochondrial ΔΨm instability on reperfusion is not triggered by mCa2+. Interestingly, inhibition of mitochondrial electron transport stabilized ΔΨm oscillations during reperfusion. The findings are consistent with mCa2+ overload being mediated by reverse-mode mNCLX activity and support ROS-induced ROS release as the primary trigger of ΔΨm instability during reperfusion injury.