Genetic recombination in all kingdoms of life initiates when helicases and nucleases process (resect) the free DNA ends to expose single-stranded (ss) DNA overhangs. Resection termination in bacteria is programmed by a DNA sequence but the mechanisms limiting resection in eukaryotes have remained elusive. Using single-molecule imaging of reconstituted human DNA repair factors, we identify a general mechanism that limits DNA resection. BLM helicase together with EXO1 and DNA2 nucleases catalyze kilobase-length DNA resection on nucleosome-coated DNA. The resulting ssDNA is rapidly bound by RPA, which is in turn phosphorylated as part of the DNA damage response (DDR). Remarkably, phosphorylated RPA (pRPA) inhibits DNA resection via regulation of BLM helicase. pRPA suppresses BLM initiation at DNA ends and promotes the intrinsic helicase strand-switching activity. These findings establish that pRPA is a critical regulator of DNA repair enzymes and provides a feedback loop between the DDR and DNA resection termination.