Excess N input results in low N use efficiency and environmental crisis, so nitrogenous fertiliser applications must be reduced. However, this can lead to low-N stress. Previous studies on low N have not explored the unique adjustment strategy to N deficiency in the short term, which is important for developing long-term N deficiency tolerance. In this case, two wheat (Triticum aestivum L.) cultivars with different tolerances to low N, Zaoyangmai (sensitive) and Yangmai158 (tolerant), were exposed to 0.25mM N as a N-deficient condition with 5.0mM N as a control. Under long-term N-deficient conditions, a significant decrease in Rubisco content resulted in decreased Rubisco activity and net photosynthetic rate (Pn) in both cultivars. However, the NO3-:soluble protein ratio decreased, and nitrate reductase and glutamine synthetase activity increased under short-term N deficiency, especially in Yangmai158. As a result, Rubisco content was not decreased in Yangmai158, while total N content decreased significantly. Moreover, increased Rubisco activase activity promoted Rubisco activation under short-term N deficiency. In sequence, Rubisco activity and Pn improved under short-term N deficiency. In conclusion, N deficiency-tolerant cultivars can efficiently assimilate N to Rubisco and enhance Rubisco activation to improve photosynthetic capabilities under short-term N deficiency conditions.