Ryder et al. report the first subcellular targeted anion transporters that accumulate in specific membrane-bound organelles to bias their natural propensity to depolarize lysosomes. Confocal microscopy revealed the ability of the naphthalimide-based transporters to localize within their intended organelles. Analogs that contained endoplasmic reticulum (ER) and lysosomal targeting motifs showed an enhanced H+/Cl− transport ability and cytotoxicity compared with non-targeted analogs. Moreover, lysosomal accumulation improved cancer cell selectivity, while ER and mitochondrial localization enhanced apoptosis in cancerous cells.