X-ray luminescence computed tomography (XLCT) is a novel molecular imaging modality that reconstructs the optical distribution of x-ray-excited phosphor particles with prior information of anatomical CT image. The prior information improves the accuracy of image reconstruction. The system can also present anatomical CT image. The optical system based on a highly sensitive charge coupled device (CCD) is perpendicular to a CT system. In the XLCT system, the x-ray was adopted to excite the phosphor of the sample and CCD camera was utilized to acquire luminescence emitted from the sample in 360-degree projections in free-space. In this study, the fluorescence diffuse optical tomography (FDOT)-like algorithm was used for image reconstruction, the structural prior information was incorporated in the reconstruction by adding a penalty term to the minimization function. The phosphor used in this study is Gd2O2S:Tb. For the simulation and experiments, the data was collected from 16 projections. The cylinder phantom was 40 mm in diameter and contains 8 mm diameter inclusion; the phosphor in the in vivo study was 5 mm in diameter at a depth of 3 mm. Both the estimation errors were no more than 5%. Based on the results from these simulation and experimental studies, the novel XLCT method has demonstrated the feasibility for in vivo animal studies.