As urban high-rise buildings increase, a mega-sub configuration is proposed by engineers, which consists of two major structural components, a megastucture as the load-bearing main structural frame and several functional substructures for residential or other usage. This paper presents a new mega-sub isolation structure, in which substructures are designed to isolate from megastructure using special isolation devices. The equations of motion of the mega-sub isolation structure are established, and its vibration mitigation mechanism is investigated systematically using a simplified lumped mass structural model. With the increase in substructure mass, working mechanism of mega-sub isolation structure is changed from tuned vibration absorber, mid-story isolation to base isolation. In the light of theoretical studies, three steel frame specimens of four mega frame stories were tested. The experimental results show that the isolated structure and lower substructure-consolidated structure perform better than aseismic structure, and transfer functions derived from the test data support the conclusion of theoretical analysis that the control mechanism of this novel mega-sub isolation structure is similar to mid-story isolated structure in the case of the mass ratio between the substructure and the megastructure closed to 1, which is the most general situation in engineering.