Walnut (Juglans regia L.) seedlings were treated with varying degrees of nitrogen (N) deficiency. The effects of N deficiency on the growth and physiology of walnut seedlings were investigated, and the adaptability and mechanism of walnut seedlings to the N-deficient environment in barren soil were explored. The purpose is to provide a scientific basis for the breeding and fertilization management of walnut varieties with high resistance and to promote the healthy and sustainable development of the walnut industry. The results showed that: (1) Under N-deficient conditions, the aboveground biomass, root biomass, chlorophyll a, chlorophyll b, and carotenoid contents of walnut seedlings were clearly lower than those of the control group, and generally diminished more obviously with the aggravation of N deficiency and the prolongation of treatment time; (2) The net photosynthetic rate, stomatal conductance, and transpiration rate of walnut seedlings in the control (CK) group were the highest overall across all N deficiency, followed by the moderate N deficiency (MN) group and the lowest in the severe N deficiency (SN) group; (3) Under N deficiency, the contents of indole-3-acetic acid (IAA), cytokinin (CTK), and abscisic acid (ABA) in the walnut seedlings increased significantly, and the contents in SN group were the highest. With processing time extended, the contents of IAA and ABA in walnut seedlings under N deficiency increased continuously, while CTK content increased rapidly within 30 d, and then declined speedily after 30 d. In addition, the ethylene content of walnut seedlings under N deficiency stress gradually increased within 30 d, and then dropped dramatically after 30 d, which was generally lower than that of the CK group; (4) During the whole experiment, the contents of putrescine (Put), spermidine (Spd), and spermine (Spm) in walnut seedlings under N deficiency stress were lower than those in the CK group in general. This indicated that the accumulation of Put, Spd, and Spm in leaves was inhibited under N deficiency; (5) Compared with the CK group, the root activity of the SN group lessened obviously, while that of MN group was slightly higher than CK group on the whole. Based on the above results, this paper concluded that under N deficiency stress conditions, walnut seedlings could strengthen root vigor by regulating hormone content, accelerating cell division and increasing root nutrient supply, thereby alleviating the stress. However, long-term N deficiency could result in the disorder of hormones and polyamine synthesis in walnut seedlings, impairing photosynthesis and slowing down root growth, thus affecting the growth of the entire seedlings and leading to seedling aging.