Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the de novo pyrimidine biosynthetic pathway. It is an attractive drug target for the treatment of various human diseases such as rheumatoid arthritis and cancers, as well as the control of certain pathogens such as Plasmodium falciparum and Helicobacter pylori. Recent studies also indicate that human DHODH inhibitors have therapeutic potential for the treatment of COVID-19. This study reports the identification of indoluidin D and its derivatives as inhibitors of human DHODH. Cell-based phenotypic screening using RIKEN NPDepo chemical library revealed that indoluidin D promoted myeloid differentiation and inhibited the proliferation of acute promyelocytic leukemia HL-60 cells. Indoluidin D also suppressed cell growth in various other types of cancer cells. Cancer cell sensitivity profiling with JFCR39 and proteomic profiling with ChemProteoBase showed that indoluidin D is a DHODH inhibitor. Indoluidin D inhibited human DHODH activity in vitro; the DHODH reaction product orotic acid rescued indoluidin D-induced cell differentiation and cell growth inhibition. Furthermore, the co-crystal structure of DHODH complexed with indoluidin D revealed that it binds in the ubiquinone-binding site of DHODH. We synthesized several indoluidin D diastereomer derivatives and demonstrated that stereochemistry was vital to their molecular activity. The indoluidin D derivative indoluidin E showed similar activity to its parent compound. Metabolome analysis of indoluidin E-treated cancer cells showed that indoluidin E markedly accumulated the DHODH reaction substrate dihydroorotic acid and its upper metabolite N-carbamoylaspartic acid. We examined the antitumor efficacy of indoluidin E in an A549 xenograft mouse model. The tumor volumes and weights in the indoluidin E-treated group were significantly lower than those in the control group. No significant body weight loss was observed at any time in the indoluidin E-treated group. Hence, indoluidin D and its derivatives selectively inhibit DHODH and suppress cancer cell growth.