Abstract: By grafting two tobacco varieties with different nitrogen efficiency, Zhongyan 100 (ZY100) and NC89, the effects of grafting techniques and different nitrogen application rates on root nitrogen absorption capacity, key nitrogen metabolism genes, related enzyme activities, and nitrogen nutrition indicators of tobacco were studied to provide theoretical understanding and technical support for regulating tobacco nitrogen absorption and utilization by grafting. The results showed that tobacco grafting altered the ability of the root system to absorb NO₃^-. Under no nitrogen (LN) application condition, the expression levels of NtNIA1, NtNIA2, NtNii2, and NtGS2 in the leaves of NC89/ZY100 tobacco plants were lower than those of NC89/NC89 plants, while the expression levels of NtGS2 in the leaves of ZY100/NC89 plants were higher than those of ZY100/ZY100 plants. The NR activity in NC89/ZY100 leaves was significantly lower than that in NC89/NC89 leaves, and the NO₃^- content in NC89/ZY100 leaves was significantly lower than that in NC89/NC89 leaves. Under the conventional nitrogen (HN) application condition, the expression levels of nitrogen metabolism genes NtNIA2, NtNii2, and NtGS1 were lower in NC89/ZY100 leaves than in NC89/NC89 leaves. The expression levels of NtNIA1 and NtGS2 in ZY100/NC89 leaves were higher than those in ZY100/ZY100 leaves. The NR activity, free amino acid content, and NO₃^- content in NC89/ZY100 leaves were significantly lower than those in NC89/NC89 leaves. The NO₃^- content in ZY100/NC89 leaves was higher than that in ZY100/ZY100 leaves. In conclusion, grafting can alter the nitrogen utilization efficiency of tobacco.