Mitochondrial calcium (Ca) uptake plays a key role in mitochondrial physiology and disease development. This process is regulated by the mitochondrial calcium uniporter (MCU) complex. DS16570511 is a membrane-permeable drug that inhibits mitochondrial Ca uptake, although its inhibitory mechanisms remain unclear. In this study, we evaluated the effects of DS16570511 on various mitochondrial functions through biochemical analyses. We found that DS16570511 affects multiple mitochondrial functions and exhibits variable potency in inhibiting individual processes. Specifically, DS16570511 not only inhibits MCU, its initially reported target, but also respiratory chain complexes and FF-adenosine triphosphatase/adenine nucleotide translocator, particularly respiratory chain complex II. Furthermore, the carboxyl group at the molecular terminus of DS16570511 plays a critical role in its inhibitory effects on mitochondrial Ca uptake through respiratory chain complex II inhibition. These findings enhance our understanding of the mechanisms by which DS16570511 inhibits mitochondrial Ca uptake and provide valuable insights for the clinical application of mitochondrial Ca uptake inhibitors.