Mitochondrial permeability transition is a phenomenon in which the mitochondrial permeability transition pore (PTP) abruptly opens, resulting in mitochondrial membrane potential (ΔΨm) dissipation, loss of ATP production, and cell death. Several genetic candidates have been proposed to form the PTP complex, however, the core component is unknown. We identified a necessary and conserved role for spastic paraplegia 7 (SPG7) in Ca2+- and ROS-induced PTP opening using RNAi-based screening. Loss of SPG7 resulted in higher mitochondrial Ca2+ retention, similar to cyclophilin D (CypD, PPIF) knockdown with sustained ΔΨm during both Ca2+ and ROS stress. Biochemical analyses revealed that the PTP is a heterooligomeric complex composed of VDAC, SPG7, and CypD. Silencing or disruption of SPG7-CypD binding prevented Ca2+- and ROS-induced ΔΨm depolarization and cell death. This study identifies an ubiquitously expressed IMM integral protein, SPG7, as a core component of the PTP at the OMM and IMM contact site. Display omitted •Mitochondrial SPG7 is essential for the PTP complex formation in multiple cell types•SPG7 interacts with CypD and VDAC at the IMM and OMM contact site•C terminus of SPG7 and CsA-binding region of CypD are necessary for PTP formation•Loss of SPG7 protects mitochondria from Ca2+- and ROS-induced PTP-dependent necrosis Using an RNAi-based screen, Shanmughapriya et al. identify mitochondrial spastic paraplegia 7 as essential for the PTP complex formation. They show that SPG7 interacts with CypD and VDAC1 at the IMM and OMM contact site, and loss of SPG7 protects mitochondria from Ca2+- and ROS-induced PTP-dependent necrosis.