Flap endonuclease-1 (FEN-1) is a structure-specific nuclease with multiple functions in DNA processing pathways. The replication and DNA repair activities of FEN-1 are critical for genomic stability in the eukaryotic cell. Through interaction with proliferation cell nuclear antigen (PCNA), FEN-1 helps coordinate Okazaki fragment maturation by removing RNA-DNA primers. FEN-1 is also required for non-homologous end joining of double-stranded DNA breaks in long patch base excision repair. The multi-functional activities of FEN-1 are regulated by various mechanisms, including protein partner interactions, post-translational modifications, and subcellular re-localization in response to cell cycle or DNA damage.