The close phylogenetic relationship between humans and nonhuman primates makes nonhuman primates an irreplaceable model for the study of human infectious diseases. In this study, we describe the development of an automatic multifunctional isolation chamber for use with medium-sized laboratory animals carrying an infectious disease. The isolator system, including the transfer chain, disinfection chain, negative air pressure isolation system, animal welfare system, and the automated system, was designed to meet all biological safety standards. To create an internal chamber environment that was completely isolated from the exterior, variable frequency drive blowers were used in the air-intake and air-exhaust system, precisely controlling the filtered airflow and providing an air-barrier protection. To transfer the material between the interior of the isolation chamber and the outside, a double door transfer port was used. A peracetic acid sterilizer and its affiliated pipeline allowed for a complete disinfection of the isolation chamber. All of the isolator system parameters can be automatically controlled by a programmable computerized menu allowing for work with different animals in different sized cages depending on the research project. The multifunctional isolator system should be a useful safety device for working with medium-sized laboratory infectious animals in high-level biosafety laboratories.