Brucellosis is a zoonotic problem worldwide, especially in developing countries and specifically in Iran, where it is endemic. This study aimed to investigate the seroprevalence of brucellosis among livestock in the 18 districts of Khuzestan Province in Southwest Iran. Serum samples of 87798 cattle and 119020 sheep were tested using the Rose Bengal Plate Test, and positive results were confirmed with serum agglutination tests (SAT) and 2-mercaptoethanol SAT (2ME-SAT). The seroprevalence derived from total samples was 0.72% for cattle and 3.01% for sheep, revealing that though brucellosis is present among livestock populations in Khuzestan and that infection among sheep is significantly higher than among cattle (P<0.05), seroprevalence in general was much lower than indicated by results of other studies of livestock in the Middle East and neighboring regions. These results nevertheless recommend implementing a policy of transparency regarding brucellosis as well as measures to effectively eradicate the disease.