To explore the association between methylation level and transcript level of Forkhead box O3a (FOXO3a) gene with ankylosing spondylitis (AS) susceptibility. Methylation levels of the FOXO3a promoter were measured in 84 AS patients and 83 healthy controls. A total of 77 patients and 66 healthy subjects were included in subsequent mRNA level testing. DNA methylation levels of 107 CpG sites on 6 CpG islands in the FOXO3a gene were investigated. This study indicated that CpG-4 and CpG-5 islands were markedly hypomethylated in AS patients. The methylation level of CpG-4 island in AS patients was negatively correlated with erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and Ankylosing Spondylitis Disease Activity Score (ASDAS). Moreover, FOXO3a mRNA levels were significantly decreased in AS patients and were obviously negatively correlated with the methylation levels of CpG-2 and CpG-5 islands in AS patients without treatment. The sensitivity and specificity of differential methylated CpG sites of FOXO3a were 74.7 and 85.4%, respectively. Besides, FOXO3a mRNA had a sensitivity of 80.0% and a specificity of 68.8%. DNA methylation and transcription of FOXO3a might be related to AS susceptibility and play a crucial role in the diagnosis of AS, but many open questions remain.