Abstract: This study aimed to explore how carbon limitation affects the carbon allocation and hydraulic characteristics of trees. It was conducted on three-year-old Robinia pseudoacacia Linn. seedlings by three-time artificial defoliation. Biomass allocation, leaf morphology, nonstructural carbohydrate (NSC) concentration in different tissues, root pressure, root hydraulic conductivity, branch water potential, branch percentage loss of conductivity (PLC), and xylem vulnerability to embolism were measured after defoliation. The biomasses of all organs, especially that of fine roots, were significantly reduced. Defoliation-induced carbon limitation significantly reduced NSC concentration in all tissues. NSC in the branch phloem, branch xylem, root phloem, and root xylem accounted for 29.6%, 20.2%, 10.2%, and 8.7% of the control, respectively. The reduction in NSC concentration in the roots was more severe than that in the branches. Root pressure and root hydraulic conductivity were also significantly reduced. Predawn and midday branch PLC was significantly increased, and xylem cavitation resistance was significantly decreased compared with that of the control. Results indicated that defoliation-induced carbon limitation reduced the ability of water uptake in the roots due to decreased biomass in the fine roots and decreased NSC. The reduced capacity of root water uptake further deteriorated hydraulic function of the roots and stems, which impeded long-distance water transport. Carbon limitation also reduced the safety of water transport by damaging cavitation resistance.