Cooperating leukemogenic events in MLL-rearranged (MLL-r) infant acute lymphoblastic leukemia (ALL) are largely unknown. We explored the role of promoter CpG island hypermethylation in the biology and therapeutic targeting of MLL-r infant ALL. The HELP (HpaII tiny fragment enrichment by ligation-mediated polymerase chain reaction [PCR]) assay was used to examine genome-wide methylation of a cohort of MLL-r infant leukemia samples (n = 5), other common childhood ALLs (n = 5), and normals (n = 5). Unsupervised analysis showed tight clustering of samples into their known biologic groups, indicating large differences in methylation patterns. Global hypermethylation was seen in the MLL-r cohort compared with both the normals and the others, with ratios of significantly (P < .001) hypermethylated to hypomethylated CpGs of 1.7 and 2.9, respectively. A subset of 7 differentially hypermethylated genes was assayed by quantitative reverse-transcription (qRT)–PCR, confirming relative silencing in 5 of 7. In cell line treatment assays with the DNA methyltransferase inhibitor (DNMTi) decitabine, MLL-r (but not MLL wild-type cell lines) showed dose- and time-dependent cytotoxicity and re-expression of 4 of the 5 silenced genes. Methylation-specific PCR (MSP) confirmed promoter hypermethylation at baseline, and a relative decrease in methylation after treatment. DNMTi may represent a novel molecularly targeted therapy for MLL-r infant ALL.