The relationship between Ala/Ser polymorphism in 133 codon of exon 3 region of the RASSF1 gene and genetic susceptibility of lung cancer in Hubei province Han population was investigated by a case-control study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was adopted to analyze the polymorphism of codon 133 of exon 3 in the RASSF1 gene of 100 pathologically diagnosed lung cancer patients, and 100 healthy controls. The relationship between different genotypes and the susceptibility of lung cancer was analyzed. Among 200 blood samples from Han people in Hubei Province, including 100 from lung cancer patients and 100 from healthy controls, the frequencies of Ala/Ala, Ala/Ser, Ser/Ser genotype of the RASSF1 in lung cancer patients were 83%, 16%, 1%, and those in healthy controls was 93%, 7%, 0% respectively, with the difference being statistically significant between two groups (P<0.05). The individuals with Ala/Ser genotype had higher risk of suffering from lung cancer, with an OR of 2.341, and 95% CI of 1.009-6.393 respectively. It was concluded that RASSF1Ala133Ser was a susceptible genetic factor of lung cancer. Ala/Ser genotype increased the risk of lung cancer.