Uncoupling protein 3 (UCP3) is known to regulate energy dissipation, proton leakage, fatty acid oxidation, and oxidative stress. To identify the putative protein regulators of UCP3, we performed yeast two hybrid screens. Here we report that UCP3 interacted with HS-1 associated protein X-1 (Hax-1), an antiapoptotic protein that was localized in the mitochondria, and is involved in cellular responses to Ca2+. The hydrophilic sequences within loop 2, and the matrix-localized hydrophilic domain of mouse UCP3, were necessary for binding to Hax-1 at the C-terminal domain, adjacent to the mitochondrial inner membrane. Interestingly, interaction of these proteins occurred in a calcium-dependent manner. Moreover, the NMR spectrum of the C-terminal domain of Hax-1 was dramatically changed by removal of Ca2+, suggesting that the C-terminal domain of Hax-1 underwent a Ca2+-induced conformational change. In the Ca2+-free state, the C-terminal Hax-1 tended to unfold, suggesting that Ca2+ binding may induce protein folding of the Hax-1 C-terminus. These results suggested that the UCP3-Hax-1 complex may regulate mitochondrial functional changes caused by mitochondrial Ca2+. (C) 2016 Elsevier Inc. All rights reserved.