Thoracic ossification of the posterior longitudinal ligament (T-OPLL) is one of the most common factors that causes thoracic spinal stenosis, resulting in intractable myelopathy and radiculopathy. Our previous study reported that the rs201153092 polymorphism present in the collagen 6A1 (COL6A1) gene was a potentially pathogenic locus for the development of T-OPLL. The present study aimed to determine whether the rs201153092 mutation causes abnormal expression of COL6A1 in Han Chinese patients with T-OPLL, and to examine the effects of this mutation on osteogenesis by establishing a model of osteogenic differentiation. COL6A1 gene mutant and wild-type mouse 3T3-E1 embryonic osteoblast models were constructed to induce the differentiation of these cells into osteoblasts. The potential of the mutation site to induce abnormal expression of the COL6A1 gene and osteogenic markers was assessed via reverse transcription-quantitative PCR and western blot analyses. The results demonstrated that the rs201153092A mutation site resulted in significantly increased COL6A1 gene expression levels in the OPLL tissues obtained following clinical surgery. This mutation was shown to play an important role in the development of T-OPLL by regulating the overexpression of the COL6A1 gene and significantly increasing the expression levels of osteogenic markers. The findings of the present study suggested that the rs201153092A mutant variant could increase the expression levels of COL6A1 and consequently play a role in the pathogenesis of T-OPLL.