Histone methyl groups can be removed by demethylases. Although LSD1 and JmjC domain-containing proteins have been identified as histone demethylases, enzymes for many histone methylation states or sites are still unknown. Here, we perform a screening of a cDNA library containing 2,500 nuclear proteins and identify hHR23A as a histone H4K20 demethylase. Overexpression of hHR23A reduces the levels of H4K20me1/2/3 in cells. In vitro, hHR23A specifically demethylates H4K20me1/2/3 and generates formaldehyde. The enzymatic activity requires Fe(II) and α-ketoglutarate as cofactors and the UBA domains of hHR23A. hHR23B, a protein homologous to hHR23A, also demethylates H4K20me1/2/3 in vitro and in vivo. We further demonstrate that hHR23A/B activate the transcription of coding genes by demethylating H4K20me1 and the transcription of repetitive elements by demethylating H4K20me3. Nuclear magnetic resonance (NMR) analyses demonstrate that an HxxxE motif in the UBA1 domain is crucial for iron binding and demethylase activity. Thus, we identify two hHR23 proteins as histone demethylases.