Understanding the oral absorption mechanisms of natural polysaccharides is critical for their further study and application. This study focused on a novel glucuronogalactomannan (STHP5) isolated from Tetrastigma hemsleyanum Diels et Gilg and systematically investigated its intestinal absorption and transcellular transport. STHP5 was fluorescently labeled with Cyanine5.5 amine (CSTHP5) and 5-(4,6-dichlorotriazinyl) aminofluorescein (FSTHP5) for in vivo and in vitro tracking. Fluorescence spectroscopy, fourier-transform infrared spectroscopy, and nuclear magnetic resonance analyses confirm successful fluorescent labeling of STHP5. Near-infrared imaging demonstrated that CSTHP5 was absorbed in the small intestine and predominantly accumulated in the liver and kidney. Single-pass intestinal perfusion confirmed preferential absorption of FSTHP5 in the ileum. In vitro, FSTHP5 uptake occurred through macropinocytosis, clathrin-mediated, and caveolae-mediated endocytosis; its intracellular trafficking was governed by the endoplasmic reticulum (ER)-Golgi and endosome-lysosome pathways with microtubule participation; and its exocytosis was predominantly mediated by the ER-Golgi pathway and microtubules. In Caco-2 cell monolayers, transcellular transport was dose- and time-dependent and likely receptor-mediated. Together, STHP5 achieves efficient oral absorption across the intestinal epithelium via an integrated transport mode involving multi-pathway endocytosis, directed intracellular trafficking, and exocytosis, providing new insights into the absorption mechanisms of hydrophilic, high-molecular-weight polysaccharides and informing delivery system design.