The mitochondrial peptidyl prolyl isomerase cyclophilin D (CypD) activates permeability transition (PT). To study the role of CypD in this process we compared the functions of brain mitochondria isolated from wild type (BM_WT) and CypD knockout (Ppif^−/−) mice (BM_KO) with and without CypD inhibitor Cyclosporin A (CsA) under normal and Ca²⁺ stress conditions. Our data demonstrate that BM_KO are characterized by higher rates of glutamate/malate-dependent oxidative phosphorylation, higher membrane potential and higher resistance to detrimental Ca²⁺ effects than BM_WT. Under the elevated Ca²⁺ and correspondingly decreased membrane potential the dose response in BM_KO shifts to higher Ca²⁺ concentrations as compared to BM_WT. However, significantly high Ca²⁺ levels result in complete loss of membrane potential in BM_KO, too. CsA diminishes the loss of membrane potential in BM_WT but has no protecting effect in BM_KO. The results are in line with the assumption that PT is regulated by CypD under the control of matrix Ca²⁺. Due to missing of CypD the BM_KO can favor PT only at high Ca²⁺ concentrations. It is concluded that CypD sensitizes the brain mitochondria to PT, and its inhibition by CsA or CypD absence improves the complex I-related mitochondrial function and increases mitochondria stability against Ca²⁺ stress.