The protective immune response against Brucella involves T-cell-mediated immunity. T-lymphocyte receptors, CD28 and cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), bind the same ligands, CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells and regulate T cell activation. CD28 delivers stimulatory signals whereas CTLA-4 provides inhibitory signals for T cell activation. Here, we investigated the association of four polymorphisms in CTLA4 (+49A/G [rs231775] and −318 C/T [rs5742909]) and its ligand CD86 (+1057 G/A [rs1129055] and +2379G/C [rs17281995]) with brucellosis infection. The study included 153 Iranian patients with active brucellosis and 128 healthy individuals as the control group. Genotyping of the CTLA4 and CD86 variants was performed using tetra amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) and PCR–restriction fragment length polymorphism analysis, respectively. It was found that the CTLA4 −318 CT genotype and T allele were present more frequently in cases than in controls and are therefore associated with an increased risk for brucellosis (−318 TT genotype; OR = 2.544, P = 0.002). Likewise, the CD86 +1057 GA and AA genotypes and A allele were associated with an increased risk of brucellosis (+1057 AA genotype; OR = 3.81, P = 0.001). However, no statistically significant difference between brucellosis patients and controls in the allele and genotype distributions of CTLA4, +49A/G (P = 0.859) and CD86, +2379G/C (P = 0.476) was found. In conclusion, CTLA4 −318 CT genotype and T allele and the CD86 +057 GA and AA genotypes and A allele play roles as risk factors for developing brucellosis infection in Iran.