Objective The forkhead transcription factor FOXP3 is a key molecular which can mediate regulatory T cells immune-related inhibitory functions. Increased levels of FOXP3-positive Tregs in peripheral blood have been proved to be associated with a less favorable prognosis in various inflammatory diseases. It is of great interest to investigate the correlation between single nucleotide polymorphism(SNP) of FOXP3 gene and the susceptibility of chronic hepatitis B(CHB). Methods Two SNPs rs2280883 and rs3761549 of FOXP3 gene in 285 patients with CHB and 295 matched controls were analyzed by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry(MALDI-TOF). Results At rs2280883, there were no significant differences in the distribution of C and T alleles between CHB donors and healthy donors, but at rs3761549, C allele was associated with CHB(P = 0.001). Compared with healthy controls, patients with CHB had high frequencies of TT genotype(73.7%) at rs2280883 or CC genotype(73.6%) at rs3761549 of FOXP3 gene. Patients who carried rs2280883 CC genotype [OR 1.744(95% CI 1.068- 2.848), P = 0.011] or rs3761549 CC genotype [OR 1.633(95% CI 1.146- 2.327), P = 0.0001] had higher risk of suffering from CHB. Stratified analysis showed that rs3761549 CC genotype was significantly associated with high incidence of HBeAg(P = 0.019). Conclusions These results suggested that FOXP3 gene polymorphism at rs2280883 and rs3761549 might be associated with the increased susceptibility to CHB.