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FTO, an N 6 -methyladenosine (m 6 A) and N 6 ,2'- O -dimethyladenosine (m 6 A m ) RNA demethylase, is a promising target for treating acute myeloid leukemia (AML) due to the significant anticancer activity of its inhibitors in preclinical models. Here, we demonstrate that the FTO inhibitor FB23-2 suppresses proliferation across both AML and CML cell lines, irrespective of FTO dependency, indicating an alternative mechanism of action. Metabolomic analysis revealed that FB23-2 induces the accumulation of dihydroorotate (DHO), a key intermediate in pyrimidine nucleotide synthesis catalyzed by human dihydroorotate dehydrogenase ( h DHODH). Notably, structural similarities between the catalytic pockets of FTO and hDHODH enabled FB23-2 to inhibit both enzymes. In contrast, the h DHODH-inactive FB23-2 analog, ZLD115, required FTO for its antiproliferative activity. Similarly, the FTO inhibitor CS2 (brequinar), known as one of the most potent h DHODH inhibitors, exhibited FTO-independent antileukemic effects. Uridine supplementation fully rescued leukemia cells from FB23-2 and CS2-induced growth inhibition, but not ZLD115, confirming the inhibition of pyrimidine synthesis as the primary mechanism of action underlying their antileukemic activity. These findings underscore the importance of considering off-target effects on h DHODH in the development of FTO inhibitors to optimize their therapeutic potential and minimize unintended consequences., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)