Mitochondrial membrane potential (MMP) is a frequently used indicator for mitochondrial function. Herein, we report a photostable near-infrared (NIR) fluorescent dye for monitoring MMP. This new probe, named NIMAP, is non-fluorescent in aqueous solution and can be activated by cell membranes, providing high fluorescence contrast and low background fluorescence. NIMAP has been validated for monitoring MMP in living mammalian cells and in mice. Due to the large fluorescence response, low fluorescence background, high photostability, and excellent tissue penetration resulting from red-shifted excitation and emission in the “optical window” above 600 nm, broad applications of this new probe are expected.