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Background and Objectives: Brucellosis is one of the most important zoonosis diseases. The aim was to estimate the seroprevalence of brucellosis in rural cattle, sheep, and goat of Bushehr Province. Methods: This cross-sectional study was conducted in 2012-2014. For the cattle population, a total of 118 villages and 891 cattle and for sheep and goat population, a total of 202 villages, 526 flocks, 3064 sheep, and 7182 goats were included. Samples were examined with Rose Bengal, 2-mercaptoethanol and wright tests. Results: In the cattle population, 28 out of 118 villages (20.34%, CI 95%: 13.94%-28.73%) and 20 out of 891 cattle (3.36%, CI 95%:2.28-4.77%) were seropositive. Additionally, in the sheep and goat population, 89 out of 202 villages (44.06%, CI 95%: 37.10%-51.20%), 142 out of 526 flocks (27%, CI 95%: 23.25%-31%), and 335 out of 10246 sheep and goats (3.27%, CI 95%: 2.93%-3.63%) were seropositive. The prevalence at the individual level of all animals was 3.53%in 2012 which was significantly higher than 2013 ( 2.59%) (P=0.02). Totally, in 3 years, the prevalence of brucellosis in sheep (3.88%) was significantly higher than goats (3%) (P = 0.02) and the prevalence in female animals (3.52%) was significantly higher than male animals (1.58%) (P