A variety of beneficial pharmacological activities have been reported for Se-enriched Grifola frondosa polysaccharides. However, little has been reported on its absorption, and its intestinal uptake and transport profiles remain unknown. Based on our previous research, the aim of this study was to investigate its absorption from two aspects - the polysaccharides and selenium of Se-enriched Grifola frondosa polysaccharides (Se-GFP-22) across Caco-2 cells in vitro. The Caco-2 cells monolayer culture model was successfully constructed to study the transport and uptake of Se-GFP-22. The results revealed that the uptake and transport of Se-GFP-22 were time- and concentration- dependent. Transport studies illustrated that Se-GFP-22 could penetrate Caco-2 cells, mainly mediated through the same routes as endocytosis and selenium in the organic selenium (Se-GFP-22) was more easily absorbed than that in the inorganic selenium control group (sodium selenite). The uptake of Se-GFP-22 may be a macropinocytosis pathway, which was an accumulation from cytoplasm to nucleus process. Se-GFP-22 was a moderately absorbed biological macromolecule testified by the apparent permeability coefficients (Papp) value and transport rates. This work illustrates the characteristics on uptake and transport of Se-GFP-22 and all these results may help to explore the mechanism of polysaccharide absorption in vitro.