Mongolia implemented a brucellosis livestock mass vaccination campaign from 2000 to 2009. However, the number of human cases did not decline since 2004 and the current epidemiological situation in Mongolia was uncertain. The objective of this study was to estimate the representative seroprevalences of humans and livestock in two provinces in view of their comparison with officially reported data. A representative cross-sectional study using cluster sampling proportional to size in humans, sheep, goats, cattle, yaks, horses, camels and dogs was undertaken to assess the apparent seroprevalence in humans and animals. A total of 8054 livestock and dog sera and 574 human sera were collected in Sukhbaatar and Zavkhan provinces. Human and animal sera were tested with the Rose Bengal and ELISA tests. The overall apparent seroprevalence of brucellosis was 27.3% in humans (95% CI 23.7–31.2%), 6.2% (95% CI 5.5–7.1%) in sheep, 5.2% (95% CI 4.4–5.9%) in goats, 16.0% (95% CI 13.7–18.7%) in cattle, 2.5% (95% CI 0.8–7.6%) in camels, 8.3 (95% CI 6.0–11.6%) in horses and 36.4% (95% CI 26.3–48.0%) in dogs. More women than men were seropositive (OR = 1.7; P < 0.0014). Human seroprevalence was not associated with small ruminant and cattle seroprevalence at the nomadic camp (hot ail) level. Annual incidence of clinical brucellosis, inferred from the seroprevalence using a catalytic model, was by a factor of 4.6 (1307/280) in Sukhbaatar and by a factor of 59 (1188/20) in Zavkhan. This represents a 15-fold underreporting of human brucellosis in Mongolia. The lack of access to brucellosis diagnostic testing at the village level hinders rural people from receiving appropriate treatment. In conclusion, this study confirms the high seroprevalence of human and livestock brucellosis in Mongolia. Stringent monitoring and quality control of operational management of a nationwide mass vaccination of small and large ruminants is warranted to assure its effectiveness. More research is needed to understand the complex animal–human interface of brucellosis transmission at different scales from farm to provincial level.