The effects of nitrogen (N) supply restriction on the CO2 assimilation and photosystem 2 (PS2) function of flag leaves were compared between two contrastive Japanese rice cultivars, a low-yield cultivar released one century ago, cv. Shirobeniya (SRB), and a recently improved high-yield cultivar, cv. Akenohoshi (AKN). Both cultivars were solution-cultured at four N supply levels from N4 (control) to N1 (the lowest). With a reduction in N-supply, contents of N (LNC), ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO), and chlorophyll (Chl) in flag leaves decreased in both cultivars. In parallel with this, the net photosynthetic rate (P-N), mesophyll conductance (g(s)), and stomatal conductance (g(s)) decreased. PN was more dominantly restricted by g(m) than g(s). The values of P-N, g(m), and RuBPCO content were larger in AKN than SRB at the four N supply levels. The content of Chl greatly decreased with N deficiency, but the reduction in the maximum quantum yield of PS2 was relatively small. Quantum yield of PS2 ((Phi(PS2)) decreased with N deficiency, and its significant cultivar difference was observed between the two cultivars at N I: a high value was found in AKN. The content ratio of Chl/RuBPCO was also significantly low in AKN. The low Chl/RuBPCO is one of the reasons why AKN maintained a comparatively high PN and (Phi(PS2) at N deficiency. The adequate ratio of N distribution between Chl and RuBPCO is the important prerequisite for the efficient and sustainable photosynthesis in a flag leaf of rice plant under low N-input.