Voltage (ΔΨ) across the inner mitochondrial membrane (IMM) controls a variety of mitochondrial function including ATP synthesis, thermogenesis, and cell death. Thus, maintenance of ΔΨ and stability of its magnitude are of a paramount significance for the physiology of the cell and the entire organism. Currently, the electron transport chain remains the only well-established mechanism for the ΔΨ maintenance. Here, we identify mitochondrial Cl⁻ channels as a crucial mechanism for the ΔΨ maintenance. We use the whole-IMM patch-clamp analysis, and demonstrate that mitochondria possess two distinct types of voltage-gated Cl⁻ channels (Cl_V), inactivating Cl_V activated at hyperpolarized voltage (hCl_V) and non-inactivating Cl_V activated at depolarized voltage (dCl_V). hCl_V is characterized by low activation threshold just below the physiological ΔΨ values and has fast inactivation. hCl_V is a novel mitochondrial Cl⁻ channel that has never been reported previously. In contrast, dCl_V activated only by profound membrane depolarization to ~ 0 mV and is completely lacking inactivation. dCl_V likely corresponds to the inner membrane anion channel or the 108-pS anion channel, but its detailed electrophysiological analysis was missing. Using optical methods and mitochondrial respiration assays, we demonstrate that mitochondrial Cl⁻ channels largely ameliorate ΔΨ depolarization induced by mitochondrial uncouplers (H⁺ leak) and Ca²⁺ uptake via the mitochondrial Ca²⁺ uniporter. Importantly, mitochondrial Cl⁻ channels profoundly delay the activation of mitochondrial permeability transition pore. Thus, hCl_V and dCl_V represent a previously unknown mechanism for ΔΨ maintenance, which could play a fundamental role in preserving mitochondrial integrity and function.