This study was conducted to describe the association between the genetic variation of the recently identified immune checkpoint molecules B7-H3 and B7-H4, and the susceptibility to ankylosing spondylitis (AS). Two single nucleotide polymorphisms (SNPs) of B7-H3 gene and three SNPs of B7-H4 gene were genotyped in 649 AS patients and 646 age- and sex-matched healthy controls. Allele, genotype frequencies and different inheritance models were performed to calculate odds ratios (ORs) and 95% confidence intervals (CIs), and the demographic and clinical parameters of patients were recorded. Our data indicated that B7-H4 rs10801935 and rs3738414 polymorphisms were correlated with AS susceptibility. In the stratification analyses, the minor A allele and GA genotype of B7-H4 rs3738414 increased the risk of AS in male patients (OR = 1.244, 95%CI = 1.026–1.508; OR = 1.453, 95%CI = 1.120–1.886, respectively). However, the association did not reach statistical significance after Bonferroni correction. Furthermore, haplotype analysis revealed that B7-H4 haplotype block TAG was a risk factor for the onset of AS (OR = 1.190, 95%CI = 1.020–1.389). These findings suggested that B7-H4 gene polymorphism may contribute to AS susceptibility in eastern Chinese Han population.