Nucleolin overexpression and DNA hypermethylation have been implicated in cancer pathogenesis, but whether and how these aberrations cooperate in controlling leukemia cell fate remains elusive. Here, we provide the first mechanistic insights into the role of nucleolin in leukemogenesis through creating a DNA hypermethylation profile in leukemia cells. We found that, in leukemia patients, nucleolin levels are significantly elevated and nucleolin overexpression strongly associates with DNMT upregulation and shorter survival. Enforced nucleolin expression augmented leukemia cell proliferation, whereas nucleolin dysfunction by RNA interference and inhibitory molecule AS1411 blocked leukemia cell clonogenic potential in vitro and impaired tumorigenesis in vivo. Mechanistic investigations showed that nucleolin directly activates NFκB signaling, and NFκB activates its downstream effector, DNA methylation machinery. Indeed, nucleolin overexpression increased NFκB phosphorylation and upregulated DNMT1 that is followed by DNA demethylation; by contrast, nucleolin dysfunction dephosphorylated NFκB and abrogated DNMT1 expression, which resulted in decreased global DNA methylation, restored p15INK4B expression and DNA hypomethylation on p15INK4B promoter. Notably, NFκB inactivation diminished, whereas NFκB overexpression enhanced DNMT1 promoter activity and endogenous DNMT1 expression. Collectively, our studies identify nucleolin as an unconventional epigenetic regulator in leukemia cells and demonstrate nucleolin-NFκB-DNMT1 axis as a new molecular pathway underlying AML leukemogenesis.