Background: The mitochondrial matrix protein cyclophilin D (CypD) is key regulator of mitochondrial function. It controls electron transport chain activity and ATP synthesis by regulating the permeability transition pore (PTP). The activity of CypD is tuned by several post-translational modifications including acetylation of lysine 166 in the mouse. Objective: To investigate how acetylation at lysine 166 modifies the ability of CypD to regulate the PTP and the ATP synthesis. Design/Methods: We generated a knock-in mouse model, were specifically in the heart, lysine 166 has been mutated into glutamine (CypDK166Q) to mimic permanent acetylation of CypD. The mice were either +/+, +/- or -/- for the expression of CypD. Calcium retention capacity (CRC) was measured with Arsenazo III in the absence or presence of cyclosporine A (CsA) or ADP. The ability of the ATP synthase to create dimers or oligomers was accessed western blotting and by the hydrolysis of ATP in an in-gel assay. Results: The CRC decreased significantly in mice when CypDK166Q mutation was expressed, while cyclosporine A or NIM811 increased the CRC. ADP increased the CRC in heart mitochondria from all mice in all experimental groups significantly (p ≤ 0.001). In-gel assays and western blotting after clear native electrophoresis show that in mitochondria from mice expressing CypD K166Q the ATP synthase is less likely to form dimers or oligomers. Conclusions: Our data show that the expression of CypD mutant of CypD increases the sensitivity to calcium and limits the generation of ATP synthase oligomers.