Precast segmental columns, as one structures, have attracted increasing attention in bridge and building construction. To understand the performance and dynamic characteristics of precast segmental columns subjected to earthquake excitations, this paper develops a novel approach for time-varying system stiffness identification of precast segmental columns under seismic excitations, based on the adaptive unscented Kalman filtering (AUKF) method. In the proposed approach, a precast segmental column model is considered as an equivalent single-degree-of-freedom (SDOF) system with time-varying system parameters. The AUKF method is employed to identify the time-varying stiffness parameters by using measured acceleration responses and then calculate dynamic displacement responses of the SDOF system to verify the accuracy of the identified stiffness parameters. Since the system parameter identification process and measurement noise covariance matrices are updated at each time step, the estimated results can be reliably obtained to evaluate the conditions of segmental columns subjected to seismic excitations. To verify the feasibility and effectiveness of using the proposed approach for time-varying system stiffness identification of segmental columns, numerical studies on a planar precast segmental column subjected to different intensities of ground motion excitations are conducted. Shake table tests on a scaled precast segmental column and a scaled monolithic column subjected to earthquake excitations are conducted in the laboratory to further verify the performance of the proposed approach. Measured acceleration responses are used for time-varying system identification. Both numerical and experimental results demonstrate that the proposed approach can successfully identify the time-varying stiffness values of precast segmental columns subjected to earthquake excitations, and the displacement responses of these columns are predicted accurately as compared with the measured ones.