Osteosarcoma (OS) is the most common malignant primary bone tumor in the world. The cytotoxic T-lymphocyte antigen-4 gene (CTLA-4) is an important candidate gene for influencing the development of OS. This study aimed to investigate the potential association of CTLA-4 genetic polymorphisms with OS risk in Chinese Han population. A total of 415 OS patients and 431 healthy controls were enrolled in this study. The created restriction site-polymerase chain reaction (CRS-PCR) and DNA sequencing methods were used to detect the genotyping of CTLA-4 c.75G[C and c.326G[A genetic polymorphisms. We observed that the genotypes/alleles of c.75G[C and c.326G[A genetic polymorphisms were statistically associated with the increased risk of OS (for c.75G[C, CC versus (vs.) GG: OR 1.72, 95% CI 1.09–2.74; C vs. G: OR 1.30, 95% CI 1.06–1.60; for c.326G[A, AA vs. GG: OR 2.12, 95% CI 1.31–3.42; A vs. G: OR 1.31, 95% CI 1.07–1.61). The allele-C/genotype-CC of c.75G[C and allele-A/genotype-AA of c.326G[A may contribute to OS susceptibility. These data indicate that CTLA-4 genetic polymorphisms are potentially related to OS risk in Chinese Han population, and might be used as molecular markers for evaluating the risk of OS.