Purpose Genetic factors play a crucial role in thoracic ossification of the ligamentum flavum (TOLF). This study aimed to better understand the association between single nucleotide polymorphisms (SNP) in functional regions of the collagen VI, alpha 1 gene (COL6A1) and TOLF, and to confirm COL6A1 as a TOLF susceptibility gene. Methods Ten tag SNPs in COL6A1 were genotyped using the SNaPshot assay, and allele and genotype frequencies were compared between TOLF patients and control individuals. The function of SNPs associated with disease was studied. For COL6A1 promoter SNPs, the transcriptional activity of each haplotype was determined by luciferase reporter assays. For COL6A1 exonic SNPs, the effect of nucleotide substitutions on COL6A1 expression was determined by western blotting. COL6A1 mRNA expression in ligamentum flavum tissues from TOLF patients with different genotypes was examined using reverse transcription real-time PCR. Results Four SNPs were associated or possibly associated with TOLF, with higher pathogenic allele and genotype frequencies seen in TOLF patients compared with controls. The rs17551710/rs7671-GG/GG genotype appeared to be related to disease severity. Nucleotide substitutions at rs17551710 and rs7671 increased COL6A1 transcriptional activity and nucleotide substitutions at rs1053312 and rs13051496 increased COL6A1 protein expression. COL6A1 mRNA expression was significantly up-regulated in individuals with rs17551710/rs7671-GG/GG and rs1053312/rs13051496-AA+AG/CC genotypes compared with other genotypes. Conclusion SNPs in the COL6A1 promoter and exonic regions are associated with TOLF in the Chinese Han population, and lead to up-regulated COL6A1 expression. We confirmed COL6A1 as a TOLF susceptibility gene that may be involved in TOLF pathology.