Abstract: To study the behavior of progressive collapse of multi-layer steel frame under different column removal scenarios, pushdown tests were carried out on two-storey two-span steel frame beam-column substructures with middle column failure and corner column failure, respectively. The resistance curves and failure modes in the collapse process were studied. The test results showed that in the middle column failure condition, the steel frame mainly relies on the bending mechanism and catenary mechanism to resist the external load, and the catenary mechanism has a certain lag. In the case of corner column failure, the steel frame mainly relies on bending mechanism to resist collapse. Furthermore, its resistance to collapse is improved due to the existence of Vierendeel mechanism. Then the finite element software ANSYS/LS-DYNA was used to simulate the specimens, and the progressive collapse analysis of six possible column removal conditions in the steel frame was carried out. By comparing the resistance curves and collapse risk of six column removal conditions structures, it can be seen that the horizontal constraint condition of steel frame is different under different column removal scenarios, so the development of Vierendeel mechanism is different. Considering the Vierendeel mechanism and bearing capacity of structure per unit area, the steel frame with penultimate corner column failure and penultimate edge column failure has the highest collapse risk.