Mutation of PHF6, which results in the X-linked mental retardation disorder Börjeson-Forssman-Lehmann syndrome, is also present in about 38% of adult T-cell acute lymphoblastic leukemias and 3% of adult acute myeloid leukemias. However, it remains to be determined exactly how PHF6 acts in vivo and what functions of PHF6 may be associated with its putative tumor suppressor function. Here, we demonstrate that PHF6 is a nucleolus, ribosomal RNA promoter-associated protein. PHF6 directly interacts with upstream binding factor (UBF) through its PHD1 domain and suppresses ribosomal RNA (rRNA) transcription by affecting the protein level of UBF. Knockdown of PHF6 impairs cell proliferation and arrests cells at G(2)/M phase, which is accompanied by an increased level of phosphorylated H2AX, indicating that PHF6 deficiency leads to the accumulation of DNA damage in the cell. We found that increased DNA damage occurs at the ribosomal DNA (rDNA) locus in PHF6-deficient cells. This effect could be reversed by knocking down UBF or overexpressing RNASE1, which removes RNA-DNA hybrids, suggesting that there is a functional link between rRNA synthesis and genomic stability at the rDNA locus. Together, these results reveal that the key function of PHF6 is involved in regulating rRNA synthesis, which may contribute to its roles in cell cycle control, genomic maintenance, and tumor suppression.