We report herein a series of rhenium(I) polypyridine complexes featuring a 2,4‐dinitrophenylsulfonamide (DNPS) unit as phosphorogenic bioimaging reagents and photocytotoxic agents. The biothiol‐selective rhenium(I) polypyridine complexes [Re(N^N)(CO)₃(py‐DNPS)](CF₃SO₃) (py‐DNPS=3‐((2,4‐dinitrophenylsulfonyl)aminomethyl)pyridine) and their DNPS‐free counterparts [Re(N^N)(CO)₃(pyridine)](CF₃SO₃) were synthesized and characterized. Upon photoexcitation, the DNPS complexes exhibited very weak luminescence as a result of photoinduced electron transfer (PET) from the excited rhenium(I) diimine moiety to the DNPS quenching unit. However, upon treatment with glutathione (GSH), the DNPS moiety was removed, resulting in emission enhancement of the solutions (I/I_o=12.6–22.2). After reaction of the DNPS complexes with GSH in living cells, intense intracellular emission and potent photocytotoxicity were both observed. Additionally, the modification of the diimine ligand with a tosylamide unit conferred on the complexes an endoplasmic reticulum (ER)‐targeting ability, which can be exploited for selective bioimaging and photocytotoxic applications.