Xylem sap primarily transports water and mineral nutrients such as nitrogen (N) from roots to shoots in vascular plants. However, it remains largely unknown how nitrogenous compounds, especially proteins in xylem sap, respond to N under- or over-supply. We found that reducing N supply increased amino-N percentage of total N in maize (Zea mays L.) xylem sap. Proteomic analysis showed that 23 proteins in the xylem sap of maize plants, including 12 newly identified ones, differentially accumulated in response to various N supplies. Fifteen of these 23 proteins were primarily involved in general abiotic or biotic stress responses, whereas the other five proteins appeared to respond largely to N under- or over-supply, suggesting distinct protein responses in maize xylem upon N under- and over-supply. Furthermore, one putative xylanase inhibitor and two putative O-glycosyl hydrolases had preferential gene expression in shoots.