Mitochondrial Ca２＋ plays an important role in the regulations of various cellular functions. Uncoupling protein ３ （UCP３） is primarily expressed in the inner membrane of skeletal muscle mitochondria. Recently, it has been reported that UCP３ is associated with Ca２＋ uptake into mitochondria. However, the mechanisms by which UCP３ regulates mitochondrial Ca２＋ uptake are not well understood. Here we report that UCP３interacts with HS‐１associated protein X‐１ （Hax‐１）, an anti-apoptotic protein that is localized in mitochondria, which is involved in cellular responses to Ca２＋. The hydrophilic sequences within the loop２, matrix-localized hydrophilic domain of mouse UCP３are necessary for binding to Hax‐１of the C-terminal domain in adjacent to mitochondrial innermembrane. Interestingly, interaction of these proteins occurs the calciumdependent manner. Moreover, NMR spectrum of the C-terminal domain of Hax‐１was dramatically changed by removal of Ca２＋, suggesting that the C-terminal domain of Hax‐１ underwent a Ca２＋-induced conformation change. In the Ca２＋-free states, C-terminal Hax‐１ didn’t change the structure, suggesting that Ca２＋ binding may induce the change of protein structure of Hax‐１ C-terminus. These studies identify a novel UCP３‐Hax‐１complex regulates the influx of Ca２＋ into mitochondria. Thus, the efficacy of UCP３‐Hax‐１in mitochondrial calcium regulation may provide a novel therapeutic approach against mitochondrial dysfunction-related disease
