In the screening of the selective inhibitors of eukaryotic DNA polymerases (pols), dehydroaltenusin was found to be an inhibitor of pol from a fungus (Alternaria tennuis). This compound inhibited only mammalian polα, and did not influence the activities of other replicative pols such as polsαs and Ƹ , but also showed no effect even on polαactivity from another vertebrate, fish, or from a plant species. Dehydroaltenusin also had no influence DNA metabolic enzymes tested. The inhibitory effect of dehydroaltenusin on mammalian polαwas dose-dependent with IC50 value of 0.5 ?M. This effect was 10-fold stronger than that of aphidicolin, a well-known potent eukaryotic polαinhibitor. The inhibitory mode of dehydroaltenusin for mammalian polαactivity was competitive with the DNA template-primer and noncompetitive with the dNTP substrate. We succeeded in chemically synthesizing dehydroaltenusin, and the compound inhibited the cell proliferation of human gastric cancer cell lines by arresting the cells at the S-phase, and preventing the incorporation of thymidine into the cells, indicating that it blocks in vivo DNA replication by inhibiting polα. This compound also induced cell apoptosis. Furthermore, we investigated in vivo anti-tumor effects on nude mice bearing solid tumors of the human cervical cancer cell line HeLa. Dehydroaltenusin was significantly effective in suppressing the growth of solid tumors, therefore, it was of interest as a candidate drug for anti-cancer treatment. These results suggested that dehydroaltenusin is a mammalian polα-specific inhibitor useful in both in vivo and in vitro experiments.