Historical masonry buildings are often heavily damaged by earthquakes, resulting in huge losses of cultural heritage. Several studies have shown the effectiveness of seismic vulnerability mitigation strategies based on base isolation systems (BISs), which allow damage to masonry buildings and their contents to be significantly reduced or even avoided. This paper presents preparatory activities and base isolation project for seismic retrofitting of an existing strategic masonry building with historical-architectural value, which is located in Perugia (Italy) and hosts the headquarters of Umbria Region, Perugia public prosecutor's office and a some offices of the Italian Ministry of Infrastructure and Transportation. The building consists of five floor levels and has an L-shaped plan with size equal to approximately 1600 m². The structure was built in the early 1950s using different materials and construction technologies. The BIS design started from a historical investigation on the original design, fabrication and modifications of the masonry building, based on archival data and available literature. An extensive investigation was carried out through on-site experimental tests on both subsoil and masonry structure, as well as geometric surveys with laser scanner technology, allowing an excellent knowledge of the building (including the development of a building information model) and the calibration of two three-dimensional structural models based on both finite element method (FEM) and equivalent frame method (EFM). The definition of the design seismic action and site amplification of seismic ground motion relied upon the outcomes of geotechnical experimental tests and site response analysis (SRA). Based on an iterative process for structural response optimization, the BIS of the existing masonry building was designed in order to minimize strengthening interventions on the building superstructure while ensuring a target period of the isolated structure lower than the resonance period highlighted by SRA. Cross-checks were carried out during the validation of the results by comparing the outputs of the FEM and EFM models. Finally, the staged fabrication of the BIS via disconnection of the fixed base structure from its foundation system and installation of isolators and rollers was carefully designed. The structural characteristics, historical value and public function of the building delineate a case study with considerable importance in the field of seismic isolation of existing masonry buildings.