This paper presents the results of a large-scale test on a three-dimensional (3D) composite floor specimen under the scenario of removed internal column. A two-bay-by two-bay substructure was 1/3 down-scaled from the prototype building due to laboratory space limitation and tested quasi-statically up to failure by using a specially designed 12-point loading system. The load-displacement responses, failure modes as well as stress development among structural components were obtained and discussed in detail. Corresponding reduced finite element (FE) models was also established afterwards and verified by comparing with test results, based on which extended analyses on the effects of reinforcement meshes, aspect ratios and decking thicknesses were studied. The efficiency of the 12-point loading system utilized in this study was also carefully examined. The experimental and computational results reveal that; (1) the ultimate loading-carrying capacity of 3D composite floor systems after the loss of a internal column is governed by the primary beam-column connections adjacent to the failed column, (2) the steel decking is the main contributor of the floor resistance (up to 65%), while the reinforcement meshes play an secondary role; (3) the 12-point loading system is an excellent substitution of uniform loads in real laboratory environments, but special attention should be paid as the the structural responses are actually underestimated by 20%.