Flap endonuclease 1 (FEN1) is a key enzyme in maintaining genomic stability and protecting against carcinogenesis. This study investigated whether functional variations in FEN1 gene are associated with DNA damage and lung cancer risk. Thirty DNA samples were sequenced to identify variants and function of the variants was examined by a set of biochemical assays. DNA damage levels were detected by comet assays in a cohort of 303 coke-oven workers and 297 controls. The association with lung cancer risk was examined in two independent case–control panels consisted of a total 1,840 lung cancer patients and 1,958 controls. We identified two single nucleotide polymorphisms (SNPs) located in the FEN1 promoter c.−69G>A (rs174538:G>A) and 3′-untranslational region c.4150G>T (rs4246215:G>T) that were associated with reduced FEN1 expression. Among coke-oven workers, DNA damage levels were significantly higher in the −69GG or GA carriers compared with the −69AA carriers. The −69GG or 4150GG carriers had a significantly increased risk for developing lung cancer compared with the −69AA or 4150TT carriers. These results highlight FEN1 as an important gene in human carcinogenesis and genetic polymorphisms in FEN1 confer susceptibility to lung cancer. Hum Mutat 30:1–9, 2009. © 2009 Wiley-Liss, Inc.