Laboratory experiments play a pivotal role in understanding the microphysical mechanisms of hailstone formation. However, few studies have applied multiple physicochemical analyses to the same hailstone, resulting in a fragmented understanding of hailstone growth. Here, for the first time, we present a comprehensive investigation of an individual layered hailstone by combining three laboratory techniques: isotopic analyses, droplet-freezing experiments and species/size distribution of insoluble particles. Isotopic analyses revealed an up-down vertical growth trajectory, with the embryo exhibiting an exceptionally low freezing temperature of -22.0°C, as considerably lower than values reported in previous studies. Droplet-freezing experiments demonstrated three to four distinct ice-nucleating particle (INP) modes, and showed that initial freezing temperatures increased by 5–6°C from the embryo toward the outer shells. The size distributions and chemical compositions of insoluble particles including organics, dust, and bioprotein particles all varied among the different INP modes. Larger insoluble particles and higher particle concentrations were associated with freezing at higher temperatures. Notably, bioprotein particles larger than 6 μm were absent from the embryo but consistently present in the outer shells, providing a mechanistic explanation for the embryo’s relatively low freezing temperature. By integrating isotopic, droplet-freezing, and insoluble particle species/size measurements on the same hailstone, this study provides new insights into the microphysical mechanisms governing hailstone growth and highlights the importance of insoluble particle characteristics in dominating hailstone ice nucleation.