A cross-sectional study was performed in Southern and Lusaka provinces of Zambia between March and September 2008 to estimate Brucella seroprevalence in cattle kept by smallholder dairy farmers (n = 185). Rose Bengal test (RBT) was used as a screening test followed by confirmation with competitive ELISA (c-ELISA). We investigated 1,323 cattle, of which 383 had a history of receiving vaccination against brucellosis and 36 had a history of abortion. Overall seroprevalence was 6.0% with areas where vaccination was practiced having low seroprevalence. Age was associated with Brucella seropositivity (P = 0.03) unlike cattle breed (P = 0.21) and sex (P = 0.32). At area level, there was a negative correlation (Corr. coeff = -0.74) between percentage of animals with brucellosis vaccination history (vaccination coverage) and level of brucellosis; percentage of animals with history of abortion (Corr. coeff. = -0.82) and brucellosis vaccination coverage. However, a positive correlation existed between brucellosis infection levels with percentage of animals having a history of abortion (Corr. coeff. = 0.72). History of vaccination against brucellosis was positively associated with a positive Brucella result on RBT (P = 0.004) whereby animals with history of vaccination against brucellosis were more likely to give a positive RBT test results (OR = 1.52). However, the results of c-ELISA were independent of history of Brucella vaccination (P = 0.149) but was positively associated with history of abortion (OR = 4.12). Our results indicate a relatively low Brucella seroprevalence in cattle from smallholder dairy farmers and that vaccination was effective in reducing cases of Brucella infections and Brucella-related abortions. Human exposure to Brucella through milk from smallholder farmers could result through milk traded on the informal market since that milk is not processed and there no quality and safety controls.