The natural resistance-associated macrophage protein 1 (Nramp1), which belongs to a conserved family of membrane metal transporters, contributes to phagocyte-autonomous antimicrobial defense mechanisms. Genetic polymorphisms in the human NRAMP1 gene predispose to susceptibility to infectious or inflammatory diseases. To characterize the transcriptional mechanisms controlling NRAMP1 expression, we previously showed that a 263 bp region upstream of the ATG drives basal promoter activity, and that a 325 bp region further upstream confers myeloid specificity and activation during differentiation of HL-60 cells induced by vitamin D. Herein, the major transcription start site was mapped in the basal region by S1 protection assay, and two cis-acting elements essential for myeloid transactivation were characterized by in vitro DNase footprinting, electrophoretic mobility shift experiments, in vivo transfection assays using linker-mutated constructs, and chromatin immunoprecipitation assays in differentiated monocytic cells. One distal cis element binds Sp1 and is required for NRAMP1 myeloid regulation. Another site in the proximal region binds CCAAT enhancer binding proteins alpha or beta and is crucial for transcription. This study implicates Sp1 and C/EBP factors in regulating the expression of the NRAMP1 gene in myeloid cells.