Abstract: Grain storage is universally recognized as one of the post-harvest infrastructure elements for national food security and societal stability. Among them, the multiple-floored grain warehouses can directly dominate the long-term reliability of the grain reserve. Its safety and stability can also require storing the grain from natural hazards and operational risks. Thereby, it is very necessary to safeguard the national food supply chains during emergencies. Particularly, the conventional single-story structures cannot fully meet the large-scale production requirements in recent years. In this study, the multi-floored grain warehouse was developed under different storage conditions. An advanced form of centralized bulk grain storage was provided for the high-density urban environments. The high mechanization was combined with efficient material handling and a compact land footprint. Thus, the storage efficiency was maximized to alleviate the land scarcity driven by rapid urbanization. A critical influencing factor under seismic loading was then obtained as the interaction between stored grain and the supporting structure in the overall dynamic response. The mass, stiffness, and inherent damping of stored grain altered the structural vibration modes, thus potentially reducing the seismic demand from the complex load transfer mechanisms. A series of shaking table experiments was conducted on the representative multi-floored grain warehouse. A 1:25 geometric similarity model was constructed to verify the measurement. Eight typical conditions of grain storage were selected: empty warehouse (EEE), fully loaded warehouse (FFF), third-floor empty (FFE), second-floor empty (FEF), first-floor empty (EFF), third-floor full (EEF), second-floor full (EFE), and first-floor full (FEE). Each condition was tested under six peak ground acceleration (PGA) levels (0.1 g, 0.2 g, 0.3 g, 0.4 g, 0.5 g, and 0.6 g). Finally, a systematic examination was carried out on the structural dynamics and seismic response. The results demonstrate that the stored grain consistently reduced the structural acceleration amplification, where the magnitude of damping was positively correlated with both PGA and storage elevation. For instance, the EFF condition decreased the top-floor acceleration by 44.1%, compared with the EEE condition at the highest test intensity (0.5 g). As such, the stored grain served as an effective vibration-mitigation medium. The peak acceleration of the grain itself was slightly lower and then delayed relative to the silo wall, indicating an energy dissipation caused by grain–grain and grain–wall friction. The displacement response was found to be jointly influenced by storage height and vertical discontinuity in grain distribution, where the latter shared the greater tendency to induce the torsional vibrations. Notably, the FEF condition generated the second-floor displacements of 24.7% and 4.0% higher than those of the EFF and FFE, respectively, under 0.5 g two-floor loading. The uneven patterns of the vertical loading significantly amplified the structural drift in the intermediate floors. Furthermore, the lateral pressure increased with the burial depth and PGA, where the strong-motion cases exhibited the pronounced overpressure. The lateral pressure at point P5 reached 1.46 times its static value in the EFE condition at 0.5 g. The substantial dynamic amplification of silo wall loads was obtained during intense earthquakes. Grain distribution also dominated the seismic performance. The optimal vertical loading can be expected to serve as a passive control strategy for the seismic demand. These findings can provide a technical basis for the seismic design and optimization of the multi-floored grain warehouses, thus supporting their application in national grain reserves and emergency supply facilities.