Background: Human brucellosis is a zoonotic disease caused by Brucella melitensis, Brucella abortus, and Brucella suis. Brucella causes a chronic disease, which subverts the immune defense system of their hosts. In this study, the prevalence of an important Brucella virulence determinant, prpA, which can modulate immune response, was determined in human isolates. Methods: Polymerase chain reaction (PCR) assay was standardized and applied to 37 isolates obtained from patient's specimens. Primers for prpA gene were designed and evaluated using bioinformatic tools. DNA sequencing was performed for further verification. Results: In the 37 Brucella isolates (31 Brucella melitensis and 6 Brucella abortus), 32 (86.4%) carried prpA gene. Conclusion: Presence of prpA gene in most isolates indicates the high prevalence of this gene among Iranian isolates and emphasizes its role in pathogenicity of this organism.