In developing countries where financial resources are limited and numerous interests compete, there is a need for quantitative data on the public health burden and costs of diseases to support intervention prioritization. This study aimed at estimating the health burden and post-exposure treatment (PET) costs of canine rabies in Ethiopia by an investigation of exposed human cases. Data on registered animal bite victims during the period of one year were collected from health centers in three districts, i.e. Bishoftu, Lemuna-bilbilo and Yabelo, to account for variation in urban highland and lowland areas. This data collection was followed by an extensive case search for unregistered victims in the same districts as the registered cases. Victims were visited and questioned on their use of PET, incurred treatment costs and the behavioral manifestations of the animal that had bitten them. Based on the collected data PET costs were evaluated by financial accounting and the health burden was estimated in Disability-Adjusted Life Years (DALYs). In total 655 animal bite cases were traced of which 96.5% was caused by dog bites. 73.6% of the biting dogs were suspected to be potentially rabid dog. Annual suspected rabid dog exposures were estimated per evaluated urban, rural highland and rural lowland district at, respectively, 135, 101 and 86 bites, which led, respectively, to about 1, 4 and 3 deaths per 100,000 population. In the same district order average costs per completed PET equaled to 23, 31 and 40 USD, which was significantly higher in rural districts. Extrapolation of the district results to the national level indicated an annual estimate of approximately 3,000 human deaths resulting in about 194,000 DALYs per year and 97,000 exposed persons requiring on average 2 million USD treatment costs per year countrywide. These estimations of the burden of rabies to the Ethiopian society provide decision makers insights into the potential benefits of implementing effective interventions.