Phosphorescence lifetime Zn2+ imaging possesses the advantage over normal fluorescence imaging in offering the more accurate temporal-spatial Zn2+ information. Herein, we report a new phosphorescent cyclometalated Ir(III) complex with a Zn2+-chelator bearing 1,10-phenanthrolin acting as ancillary ligand, Zin-IrDPA, which displays the specific Zn2+-induced enhancement of phosphorescence and phosphorescence lifetime, and the mitochondria-targeting ability. Moreover, its Zn2+-induced phosphorescence lifetime enhancement factor is not affected by medium lipophilicity, viscosity, polarity, and especially ambient oxygen. The reversible tracking of introduced exogenous labile Zn2+ in MCF-7 and HeLa cells via phosphorescence imaging and phosphorescence lifetime imaging (PLIM) have been realized with Zin-IrDPA. Moreover, PLIM with Zin-IrDPA is able to track the SNOC-stimulated endogenous Zn2+ release in mitochondria.