Ribonucleotide reductase (RNR) is an attractive target for anticancer agents given its central function in DNA synthesis, growth, metastasis, and drug resistance of cancer cells. The current clinically established RNR inhibitors have the shortcomings of short half-life, drug resistance, and iron chelation. Here, we report the development of a novel class of effective RNR inhibitors addressing these issues. A novel ligand-binding pocket on the RNR small subunit (RRM2) near the C-terminal tail was proposed by computer modeling and verified by site-directed mutagenesis and nuclear magnetic resonance (NMR) techniques. A compound targeting this pocket was identified by virtual screening of the National Cancer Institute (NCI) diverse small-molecule database. By lead optimization, we developed the novel RNR inhibitor COH29 that acted as a potent inhibitor of both recombinant and cellular human RNR enzymes. COH29 overcame hydroxyurea and gemcitabine resistance in cancer cells. It effectively inhibited proliferation of most cell lines in the NCI 60 human cancer panel, most notably ovarian cancer and leukemia, but exerted little effect on normal fibroblasts or endothelial cells. In mouse xenograft models of human cancer, COH29 treatment reduced tumor growth compared with vehicle. Site-directed mutagenesis, NMR, and surface plasmon resonance biosensor studies confirmed COH29 binding to the proposed ligand-binding pocket and offered evidence for assembly blockade of the RRM1-RRM2 quaternary structure. Our findings offer preclinical validation of COH29 as a promising new class of RNR inhibitors with a new mechanism of inhibition, with broad potential for improved treatment of human cancer.