Background: Brucellosis is an important zoonotic disease in developing countries yet it is often not recognized, goes unreported and does not attract public health action by these governments including Uganda. Objective: To estimate the sero-prevalence and assess modifiable risk factors associated with Brucella seropositivity in cattle keepers and consumers of unpasteurized milk in Uganda. Methods: One group comprised of 161 individuals randomly selected from households living on farms that had Brucella sero-positive cattle and/or goats in Mbarara District from an earlier survey; the second group comprised of 168 randomly selected individuals attending an HIV voluntary counseling and testing clinic in Kampala District. Sera samples were tested using Rapid Plate Agglutination Test, Standard Tube Agglutination Test and cELISA. Results: The sero-prevalence of brucellosis among exposed cattle keepers in Mbarara and consumers of unpasteurised milk in Kampala Districts was 5.8% (95%CI: 3.3%, 8.3%) and 9% (95%CI: 13.3%, 4.7%), respectively. Consumption of unboiled milk was significantly (p=0.004) associated with seropositivity in Mbarara District. There was no association between sero- positivity with age, sex and awareness of human brucellosis. Conclusion: Human brucellosis is prevalent among livestock rearing communities and consumers of unpasteurised milk. The continued consumption of unboiled milk is a major health risk.