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Decreasing nitrogen (N) supply affected the normal growth of <i>Oryza sativa</i> (<i>O. sativa</i>) seedlings, reducing CO₂ assimilation, stomatal conductance (gs), the contents of chlorophylls (Chl) and the ratio of Chl <i>a</i>/Chl <i>b</i>, but increasing the intercellular CO₂ concentration. Polyphasic chlorophyll <i>a</i> fluorescence transient and relative fluorescence parameters (JIP test) results indicated that N deficiency increased F_o, but decreased the maximum quantum yield of primary photochemistry (F_v/F_m) and the maximum of the IP_phase, implying that N-limiting condition impaired the whole photo electron transport chain from the donor side of photosystem II (PSII) to the end acceptor side of PSI in <i>O. sativa</i>. N deficiency enhanced the activities of the antioxidant enzymes, such as ascorbate peroxidase (APX), guaiacol peroxidase (GuPX), dehydro–ascorbate reductase (DHAR), superoxide dismutase (SOD), glutathione peroxidase (GlPX), glutathione reductase (GR), glutathione S-transferase (GST) and <i>O</i>-acetylserine (thiol) lyase (OASTL), and the contents of antioxidant compounds including reduced glutathione (GSH), total glutathione (GSH+GSSG) and non-protein thiol compounds in <i>O. sativa</i> leaves. In contrast, the enhanced activities of catalase (CAT), DHAR, GR, GST and OASTL, the enhanced ASC–GSH cycle and content of sulfur-containing compounds might provide protective roles against oxidative stress in <i>O. sativa</i> roots under N-limiting conditions. Quantitative real-time PCR (qRT-PCR) analysis indicated that 70% of the enzymes have a consistence between the gene expression pattern and the dynamic of enzyme activity in <i>O. sativa</i> leaves under different N supplies, whereas only 60% of the enzymes have a consistence in <i>O. sativa</i> roots. Our results suggested that the antioxidant system and sulfur metabolism take part in the response of N limiting condition in <i>O. sativa</i>, and this response was different between leaves and roots. Future work should focus on the responsive mechanisms underlying the metabolism of sulfur-containing compounds in <i>O. sativa</i> under nutrient deficient especially N-limiting conditions.