Ammonium (NH4 +) is the preferred nitrogen nutrient over nitrate (NO3 –) in Oryza sativa L. (rice), but photosynthetic capacity is enhanced by partial NO3 – nutrition (PNN). The role of cytokinin in the effects of PNN on photosynthetic capacity is unknown. We investigated effects of PNN on six cytokinin fractions in roots, xylem sap, and leaves and on the expression of eight cytokinin synthesis genes in the roots of Nanguang and Elio rice cultivars. The effect of exogenous cytokinin (6-BA) on leaf growth and photosynthetic activity was examined. Cell expansion and CO2 assimilation in the first fully expanded leaf were enhanced by PNN in Nanguang but not in Elio. The concentrations of cytokinins in roots, xylem sap, and leaves of Nanguang increased approximately 25–34 % with PNN compared with sole NH4 +, but no difference was observed in Elio. Exogenous 6-BA counteracted the effects of sole NH4 + on leaf growth and photosynthetic activity in both cultivars. OsIPT3 was the key NO3 –-responsive cytokinin synthesis gene in cv. Nanguang. High NO3 – responsiveness is associated with increased cytokinin synthesis and transport from the root to the leaf and is strongly related to a higher photosynthetic capacity in cv. Nanguang.