Rice is one of the most susceptible plant to iron (Fe) deficiency under neutral and alkaline conditions. Alkaline stress induces H2O2 production and increases the deposition of Fe on roots surface, which causes leaf chlorosis and Fe deficiency in rice. Gene chip and qRT-PCR analysis indicated that the expression of nitrate reductase (NR) genes were down-regulated by alkaline treatment, which resulted in significantly decreased nitrate activities and nitric oxide (NO) production in epidermis and stele, where the H2O2 was accumulated. In contrast, treatment with sodium nitroprusside (SNP), a NO donor, strongly alleviates alkaline-induced Fe deficiency by limiting Fe plaque formation. Increasing the NO signal significantly reduces the accumulation of H2O2 and lignin barrier, but enhances phenolic acid secretion in root epidermis and stele under alkaline stress. The secreted phenolic acid effectively mobilized the apoplast Fe and increased Fe uptake in root, thus which alleviate the Fe deficiency response and down-regulate expression of Fe uptake genes under alkaline condition. In conclusion, alkaline stress inhibits the NR activity and NO production in roots of rice, which plays a vital role in mobilizing the apoplast Fe by regulation of H2O2 and phenolic acids concentrations.