Aberrant ribosome biogenesis and enlarged nucleoli have long been used by pathologists as a marker of aggressive tumors. Suppression of RNA polymerase I (Pol I) transcription machinery within the nucleolus could be a direct way to trigger the nucleolar stress and to inhibit the rapid proliferation of cancer cells. Here we modified cisplatin with an analogue of the selective inhibitor of RNA polymerase I-mediated transcription BMH-21 to develop a novel platinum-based Pol I selective inhibitor. We show that this novel monofunctional platinum-based agent, P1-B1, had enhanced antitumor activity of up to 17-fold greater than the clinical drug cisplatin in cisplatin-resistant non-small cell lung cancer cells. P1-B1 also had significantly lower cytotoxicity compared to cisplatin as well as the Pol I selective inhibitor BMH-21 in MRC-5 normal lung fibroblast cells, and the selectivity index (SI) greatly increases. Mechanistic investigations revealed that P1-B1 displayed significant nucleolar accumulation, selectively inhibited Pol I transcription, and induced nucleolar stress, leading to S-phase arrest and apoptosis. Our results suggest that the effects of P1-B1 are mechanistically distinct from those of conventional platinum agents and the recently described non-classical platinum compounds and that functionalizing platinum-based agents with directly Pol I transcription inhibition properties may represent an improved modality for cancer treatment.