Q fever is a widespread zoonosis that is caused by Coxiella burnetii (C. burnetii), and ruminants are identified as the main sources of human infections. Some human cases have been described, but very limited information was available about Q fever in ruminants on Reunion Island, a tropical island in the Indian Ocean. A cross-sectional study was undertaken from March 2011 to August 2012 to assess the Q fever prevalence and to identify the major risk factors of C. burnetii infection in ruminants. A total of 516 ruminants (245 cattle, 137 sheep and 134 goats) belonging to 71 farms and localized in different ecosystems of the island were randomly selected. Samples of blood, vaginal mucus and milk were concomitantly collected from females, and a questionnaire was submitted to the farmers. Ticks from positively detected farms were also collected. The overall seropositivity was 11.8% in cattle, 1.4% in sheep and 13.4% in goats. C. burnetii DNA was detected by PCR in 0.81%, 4.4% and 20.1% in cow, sheep and goat vaginal swabs, respectively. C. burnetii shedding in milk was observed in 1% of cows, 0% in sheep and 4.7% in goats. None of the ticks were detected to be positive for C. burnetii. C. burnetii infection increased when the farm was exposed to prevailing winds and when there were no specific precautions for a visitor before entering the farm, and they decreased when a proper quarantine was set up for any introduction of a new ruminant and when the animals returned to the farm at night. MLVA genotyping confirmed the role of these risk factors in infection.