The GFI1 gene encodes a transcriptional repressor, which regulates myeloid differentiation. In the mouse, Gfi1 deficiency causes neutropenia and an accumulation of granulomonocytic precursor cells that is reminiscent of a myelodysplastic syndrome. We report here that a variant allele of GFI1 (GFI136N) is associated with acute myeloid leukemia (AML) in white subjects with an odds ratio of 1.6 (P < 8 × 10−5). The GFI136N variant occurred in 1806 AML patients with an allele frequency of 0.055 compared with 0.035 in 1691 healthy control patients in 2 independent cohorts. We observed that both GFI1 variants maintain the same activity as transcriptional repressors but differ in their regulation by the AML1/ETO (RUNX1/RUNX1T1) fusion protein produced in AML patients with a t(8;21) translocation. AML1/ETO interacts and colocalizes with the more common GFI136S form in the nucleus and inhibits its repressor activity. However, the variant GFI136N protein has a different subnuclear localization than GFI136S. As a consequence, AML1/ETO does not colocalize with GFI136N and is unable to inhibit its repressor activity. We conclude that both variants of GFI1 differ in their ability to be regulated by interacting proteins and that the GFI136N variant form exhibits distinct biochemical features that may confer a predisposition to AML.