Fragile histidine triad (FHIT) is a suppressor gene related to cervical cancer through CpG islandhypermethylation. Folate is a water-soluble B-vitamin and an important cofactor in one-carbon metabolism. Itmay play an essential role in cervical lesions through effects on DNA methylation. The purpose of this study wasto observe effects of folate and FHIT methylation and HPV 16 on cervical cancer progression. In this study, DNAmethylation of FHIT, serum folate level and HPV16 status were measured using methylation-specific polymerasechain reaction (MSP), radioimmunoassay (RIA) and polymerase chain reaction (PCR), respectively, in 310 womenwith a diagnosis of normal cervix (NC, n=109), cervical intraepithelial neoplasia (CIN, n=101) and squamous cellcarcinoma of the cervix (SCC, n=101). There were significant differences in HPV16 status (χ2=36.64, P<0.001),CpG island methylation of FHIT (χ2=71.31, P<0.001) and serum folate level (F=4.57, P=0.011) across the cervicalhistologic groups. Interaction analysis showed that the ORs only with FHIT methylation (OR=11.47) or onlywith HPV 16 positive (OR=4.63) or with serum folate level lower than 3.19ng/ml (OR=1.68) in SCC group wereall higher than the control status of HPV 16 negative and FHIT unmethylation and serum folate level more than3.19ng/ml (OR=1). The ORs only with HPV 16 positive (OR=2.58) or with serum folate level lower than 3.19ng/ml (OR=1.28) in CIN group were all higher than the control status, but the OR only with FHIT methylation(OR=0.53) in CIN group was lower than the control status. HPV 16 positivity was associated with a 7.60-foldincreased risk of SCC with folate deficiency and with a 1.84-fold increased risk of CIN. The patients with FHITmethylation and folate deficiency or with FHIT methylation and HPV 16 positive were SCC or CIN, and thepatients with HPV 16 positive and FHIT methylation and folate deficiency were all SCC. In conclusion, HPV16 infection, FHIT methylation and folate deficiency might promote cervical cancer progression. This suggeststhat FHIT may be an effective target for prevention and treatment of cervical cancer.