In traditional Chinese medicine (TCM), Gardeniae Fructus is classified as a “cold” herb, a property that is increasingly explained by modern research showing that it can alleviate related disorders through modulation of the central nervous and endocrine systems, energy metabolism, and gut microbiota. This study aimed to elucidate the material foundation and biological mechanisms underlying its cold property. Chemical components of Gardeniae Fructus were separated via multi-stage extraction and characterized by GC-MS and LC-MS, yielding four distinct fractions: aliphatic, iridoid, crocin, and polysaccharide. In a rat model of heat syndrome induced by levothyroxine sodium, administration of the total extract or individual fractions over 15 days modulated central nervous, endocrine, and energy metabolism indicators, with the iridoid, crocin, and polysaccharide fractions demonstrating significant cold properties. Gut microbiota analysis revealed that the total extract, polysaccharide, and iridoid fractions notably reshaped microbial structure, reducing Firmicutes and Lactobacillus abundance. These findings indicate that the iridoid, crocin, and polysaccharide fractions may be key material bases for the cold property of Gardeniae Fructus, with the iridoid fraction exhibiting the strongest effect.