Summary Sap molecules are transported by xylem flow throughout the whole plant body. Factors regulating the xylem transport of different molecules remain to be identified. We used fluorophores to visualize xylem transport from roots to leaves in Arabidopsis thaliana. Several previously established Arabidopsis lines with modified xylem cell walls were used to determine the contribution of xylem cell walls to xylem transport. Fluorophores underwent xylem flow‐dependent transport from roots to leaves within 20 min. A comparison of rhodamine, fluorescein and three fluorescently labeled CLV3/ESR‐related (CLE) peptides revealed cargo‐dependent xylem transport patterns in terms of leaf position and vein order. Only minor changes in amino acid sequence were sufficient to alter the xylem transport patterns of the labeled CLE peptides. We found that the xylem transport pattern of fluorescein was affected in Arabidopsis lines with modified AtXYN1, LAC4 or CCoAOMT1 expression. In these lines, application of a defense inducer, pipecolic acid, to roots resulted in altered defense response patterns in leaves, whereas all the lines showed wild‐type‐like responses when pipecolic acid was sprayed onto leaves. The combined results reveal a finely controlled cargo‐dependent xylem transport and suggest that the xylem cell wall structure is crucial for this transport system.