As a DNA repair protein, Flap endonuclease 1 (FEN1) plays crucial parts in preventing carcinogenesis. Two functional germ line variants (-69G > A and 4150G > T) in the FEN1 gene have been associated with DNA damage levels in coke oven workers and lung cancer risk in general populations. However, the role of these genetic variants on gastrointestinal cancer susceptibility is unknown. Therefore, we evaluated the association between these polymorphisms and gastrointestinal cancer risk in two independent case-control cohorts consisted of a total of 1850 gastrointestinal cancer (hepatocellular carcinoma, esophageal cancer, gastric cancer and colorectal cancer) patients and 2222 healthy controls. The impact of these variations on FEN1 expression was also examined using liver, esophagus, stomach and colon normal tissues. It was found that the FEN1 -69GG genotypes were significantly correlated to increased risk for developing gastrointestinal cancer compared with the -69AA genotype in both cohorts [Jinan cohort: odds ratios (OR) = 2.14, 95% confidence interval (CI) = 1.47-2.80, P = 1.0 × 10(-)(6); Huaian cohort: OR = 1.93, 95% CI = 1.37-2.50, P = 0.5 × 10(-6)]. Similar results were observed for 4150G > T polymorphism. In the combined meta-analyses, OR for -69GG or 4150GG genotype was 2.02 (95% CI = 1.59-2.45) or 1.86 (95% CI = 1.45-2.28) compared with -69AA or 4150TT genotype. In vivo FEN1 messenger RNA expression analyses showed that the -69G or 4150G allele carriers had ∼2-fold decreased FEN1 expression in gastrointestinal tissues compared with -69A or 4150T carriers, indicating that lower FEN1 expression may lead to higher risk for malignant transformation of gastrointestinal cells. Our results highlight FEN1 as an important gene in human gastrointestinal oncogenesis and genetic polymorphisms in FEN1 confer susceptibility to gastrointestinal cancers.