Rabies is an important causative agent of disease resulting in an acute infection of the nervous system and death of the individual. Rabies remains an important public health program in developing countries, and the indigenous threat of rabies continues in developed countries because of wildlife reservoirs. Globally, there are about 55,000 fatal human cases of rabies each year [WHO, 2007]. Control of rabies in wildlife remains an important challenge for government offices. There are numbers of rabies vaccines commercially available for controls of wildlife rabies. However, these vaccines currently distributed to wildlife do not effectively immunize all at-risk species, especially skunks. Alternative efficacious vaccines are needed. A human adenovirus rabies glycoprotein recombinant vaccine candidate (AdRG1.3), developed by the Rabies Research and Development Unit at Ontario Ministry of Natural Resources, Canada, has shown the most promising result in laboratory trials. The adenovirus used in rabies vaccine laden bait is produced using HEK 293 cell culture process. This presentation will focus on demonstrating the successful scale-up of AdRG1.3 adenovirus production from 1 liter to 500 liter to manufacture large quantities of bulk material required to support field trials and demonstrate efficacy of this new vaccine. The robustness of production process was improved through elimination of medium replacement operation at the time of virus infection, and culture titer was increased by over 3 folds through optimization of cell culture medium. The elimination of medium replacement step reduced the risk of culture contamination, and resulted in significant saving in material expenses and reduction in labor costs. Over 10,000 liters of active AdRG1.3 adenovirus cultures were manufactured so far to support field trials. AdRG1.3 adenovirus is formulated and packaged in baits using Artemis Technologies Inc. proprietary technology prior to aerial-baiting. AdRG1.3 rabies vaccine has been distributed by several provincial agencies to testing areas located in Ontario, Quebec and New Brunswick provinces, Canada, for field trials in yearly campaigns from 2006 to 2009. The field results showed that the new vaccine was more efficient than the existing ones in immunizing animals that were previously difficult to vaccinate.