Post-anthesis nitrogen uptake and translocation play critical roles in photosynthetic assimilation and grain filling. However, their effects on leaf stay-green characteristics, dry matter accumulation, and translocation after anthesis remain unclear. In this study, post-anthesis N uptake and translocation between two different rice genotypes (Yongyou12 and Zhongzheyou1) were compared through soil nitrogen leaching treatments at the meiosis stage (MST) and anthesis stage(AST) respectively, and their effects on leaf stay-green duration, photosynthesis, dry matter accumulation and translocation during ripening and yield formation were estimated. The results showed that the soil nitrate-N and ammonium-N contents in Yongyou12 pots decreased significantly, and post-anthesis N uptake was 2.0–3.4 fold higher in Yongyou12 than in Zhongzheyou1. The activities of N-metabolism enzymes and antioxidant enzymes were higher, and flag-leaf photosynthesis and dry matter accumulation during ripening were greater, in Yongyou12 than in Zhongzheyou1. However, insufficient available soil N led to significant decreases in the activities of N- metabolism enzymes, decreased flag-leaf photosynthesis, increased translocation of dry matter and N pre-anthesis, accelerated leaf senescence, shorter duration of the leaf stay-green period, and decreased dry matter accumulation and grain plumpness. In addition, the effect of N uptake after anthesis on yield is greater for rice genotypes that depend on post-anthesis dry matter accumulation and an expanded sink capacity.