Isolation technique has been acceded as a part of the China Seismic Code for Design of Buildings. In this code, the limitations for using isolation design are very strict, superstructure must be regular and the isolation layer must be located on the top of base (base isolated structure). Because of the needs of architecture and function or the feasibility of technique, some limitations have been broken in recent projects. Sometimes isolated layer can be set on the intermediate story, so-called the mid-story isolated structure. According to the characteristic of structure, isolation layer of mid-story isolated structure is set on a place where the structure’s vertical stiffness is suddenly changed, as like the top of the first story, middle story, conversion story of the structure. Laminated rubber bearings (LRB) are adopted as an isolation layer. Because the isolation layer is set on intermediate story, the whole structure is divided into superstructure and substructure; the structure’s dynamic characteristics are changed. The mechanism of mid-story isolated structure appears different characteristic compared with base isolation. The aim of mid-story isolation is not only to reduce seismic responses of superstructure, but also to reduce seismic responses of the substructure. Theoretical analysis and the shaking table test of the mid-story isolated structure were carried. And the response of mid-story isolated structure is discussed by comparing with the response of base-isolated structure and base fixed structure. The key problems of mid-story isolated structure are the force condition and the interaction of the structure up and below the isolation layer. Many factors, such as the number of story, mass, stiffness of superstructure and substructure, parameter of the isolation layer, have influence on the seismic behavior of the mid-story isolated structure. The optimum combination relationship of these factors is presented and dynamic characteristics and dynamic responses are investigated.