Tim17 and Tim23 are the main subunits of the TIM23 complex, one of the two major essential mitochondrial inner-membrane protein translocon machineries (TIMs). No chemical probes that specifically inhibit TIM23-dependent protein import were known to exist. Here we show that the natural product stendomycin, produced by Streptomyces hygroscopicus, is a potent and specific inhibitor of the TIM23 complex in yeast and mammalian cells. Furthermore, stendomycin-mediated blockage of the TIM23 complex does not alter normal processing of the major regulatory mitophagy kinase PINK1, but TIM23 is required to stabilize PINK1 on the outside of mitochondria to initiate mitophagy upon membrane depolarization.