The cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and costimulatory molecule (CD80/CD86) genes are important susceptibility genes associated with autoimmune diseases. CTLA-4 polymorphisms have been found to be associated with various autoimmune diseases. However, the association data are inconsistent for rheumatoid arthritis (RA). We investigated the genetic association of CTLA-4 and CD86 polymorphisms with RA in a Chinese population. Four single nucleotide polymorphisms (SNPs) (rs5742909 and rs231775 in CTLA-4, and rs17281995 and rs1129055 in CD86) were genotyped in 213 patients with RA and 303 healthy controls using polymerase chain reaction-restriction fragment length polymorphism. The genotype and allele distributions of rs5742909 differ significantly between RA patients and controls (P < 0.05) and the dominant model was found to be the best inheritance model. Stratification studies showed that CTLA-4 gene polymorphisms were more significantly associated with rheumatoid factor-negative and anti-cyclic citrullinated peptide-negative subgroups in the southeastern Han Chinese population. We also found that the haplotype of 2 CTLA-4 SNPs showed significant association with the disease (P = 0.0025) with the T-A (OR = 1.88, 95%CI = 1.12-3.15) and T-G (OR = 3.45, 95%CI = 1.10-10.87) haplotypes being observed more frequently in cases than in controls. We failed to find any significant association of the 2 CD86 SNPs with RA. These results indicate that the polymorphisms of CTLA-4 (rs5742909) may be important genetic factors for RA risk in the southeastern Han Chinese population.