Abstract: The safe storage of grain is universally recognized as a fundamental safeguard for national food security and societal stability. As a core element of post-harvest grain storage infrastructure, the safety and stability of multi-floored grain warehouses directly influence the long-term reliability of the grain reserve system. These warehouses ensure stored grain remains protected from natural hazards and operational risks, thereby safeguarding the continuity of national food supply chains during emergencies. Developed from traditional single-storey structures, multi-floored designs provide an advanced form of centralized bulk grain storage for high-density urban environments. They combine high mechanization with efficient material handling systems and a compact land footprint, thus maximizing storage efficiency and alleviating land scarcity driven by rapid urbanization. Under seismic loading, the interaction between stored grain and the supporting structure becomes a critical factor in the overall dynamic response. The mass, stiffness, and inherent damping characteristics of stored grain alter the structural vibration modes, potentially reducing seismic demand but also introducing complex load transfer mechanisms. To investigate these effects, a series of shaking table experiments were conducted using a 1:25 geometric similarity model of a representative multi-floored grain warehouse. Eight typical grain storage conditions were analyzed: 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, 0.2, 0.3, 0.4, 0.5 and 0.6 g) to systematically examine structural dynamic characteristics and seismic response mechanisms. The results demonstrate that stored grain consistently reduces structural acceleration amplification, with the magnitude of damping positively correlated with both PGA and storage elevation. For instance, the top-floor acceleration under the EFF condition decreased by 44.1% compared to the EEE condition, highlighting the potential of stored grain to serve as an effective vibration-mitigation medium. The peak acceleration of the grain itself was slightly lower and delayed relative to the silo wall, indicating an energy dissipation process caused by grain–grain and grain–wall frictional interactions. The displacement response was found to be jointly influenced by storage height and vertical discontinuity in grain distribution, with the latter having a greater tendency to induce torsional vibrations. Notably, under 0.5 g two-floor loading, the FEF condition generated second-floor displacements 24.7% and 4.0% higher than that of EFF and FFE, respectively, suggesting that uneven vertical loading patterns can significantly amplify structural drift in intermediate floors. Furthermore, lateral pressure increased with burial depth and PGA, with strong-motion cases exhibiting pronounced overpressure effects. In the EFE condition at 0.5 g, the lateral pressure at point P5 reached 1.46 times its static value, indicating substantial dynamic amplification of silo wall loads during intense earthquakes. These findings clarify the mechanisms by which grain distribution affects seismic performance and suggest that optimizing vertical loading can function as a passive control strategy to reduce seismic demand. The results provide a technical basis for the seismic design and optimization of multi-floored grain warehouses, supporting their application in national grain reserves and emergency supply facilities.