The use of mixed nitrate and ammonium supply (mixed-N) as a nitrogen source can improve plant growth. Phytohormones in the xylem sap are involved in root-shoot signaling and regulation of plant growth and development. However, the role of xylem sap phytohormones in the shoot growth with mixed-N supply is poorly understood. Here, pot-grown maize seedlings fed with different nitrogen forms (NO 3 −, NH 4 +, and mixed-N) at 50, 100, 200, and 400 mg N kg −1 soil (N 50, N 100, N 200, and N 400, respectively) were used to measure shoot biomass, stomatal conductance, leaf water saturation deficit (LWSD), and concentrations of zeatin riboside (ZR), auxin (IAA) and abscisic acid (ABA) and their concentration ratios in the xylem sap. Results showed that mixed-N supply increased maize shoot biomass and LWSD compared with NO 3 − or NH 4 + applied alone regardless of the N rates. Mixed-N supply significantly increased ZR and IAA concentrations, and decreased ABA concentration (thus increased the ZR/ABA, IAA/ABA, and (ZR+IAA)/ABA concentration ratios), which were correlated positively with shoot growth and LWSD. And the stomatal conductance showed a negative correlation with LWSD under different N forms and rates. Our results indicated that mixed-N supply could facilitate maize shoot growth by regulating phytohormones and their concentration ratios in the xylem sap to control stomatal function and improve leaf hydration status.