The transmissibility-based semi-active (TSA) controller was developed in the existing study by the authors, which can effectively enhance the performance of base-isolated buildings under both strong long- and short-period earthquake ground motions. Since the performance of the TSA controller was only evaluated with numerical simulation in the existing study, this paper further validates its performance experimentally by conducting real-time hybrid simulation (RTHS). A three-story base isolated building was designed based on a simplified design procedure, where the base isolation system of the building consisted of three different devices, that is, a magneto-rheological (MR) damper, rubber bearing, and linear bearings. The base isolation system was experimentally tested with the MR damper controlled by the TSA controller, and the building superstructure was analytically modeled. It was shown that the TSA controller makes the system damping high under long-period ground motions and low under short-period ground motions, which performed uniquely as intended. As a result, the isolator displacement was effectively reduced under long-period ground motions, while the story drift and acceleration responses were also reduced under short-period ground motions, all of which are difficult to achieve at the same time using passive damping only.