Brucellosis remains an endemic disease in livestock populations in Rwanda, but the prevalence of the disease varies by geographic region. The common use of informal milk marketing channels represents a health hazard to humans when milk from Brucella-infected cows from one or more households is mixed with milk from other households for human consumption. In Rwanda, knowledge about the burden of brucellosis in livestock and factors associated with farmers' choice of milk marketing channels is very limited. The objectives of this study were 1) to estimate the seroprevalence of brucellosis in livestock in Rwamagana District in Rwanda and 2) to estimate the frequency of and identify determinants associated with farmers' choice of milk marketing channels. The seroprevalence of brucellosis in livestock at the household level was 1/160 (0.6%; 95% CI = 0.1-3.4%). Among 100 households that sold milk, 72 used informal milk trade channels. By the use of logistic regression, milk price and distance to closest milk collection center were positively associated with the use of informal milk marketing channels. Although the seroprevalence of brucellosis in livestock in Rwamagana District was low, the risk of disease transmission to humans through consumption of unpasteurized milk or milk products is not negligible. In this report, we present several policy options available to animal health authorities that can mitigate the risk of brucellosis disease transmission in populations.