Iron (Fe) is an essential element for plant growth, development, and metabolism. Due to its lack of solubility and low bioavailability in soil, Fe levels are usually far below the optimum amount for most plants’ growth and development. In apple production, excessive use of nitrogen fertilizer may cause iron chlorosis symptoms in the newly growing leaves, but the regulatory mechanism is unclear. In this study, it was found that low nitrate (NO3-, LN) application could alleviate the symptoms of Fe deficiency, LN treatment promoted lower the rhizosphere pH, which was beneficial for root Fe acquisition, meanwhile, LN treatment increased citrate and abscisic acid (ABA) accumulation in root, which promoted Fe transport from root to shoot and maintained Fe homeostasis. Moreover, RNA-Seq and qRT-PCR analysis showed that nitrate application caused differential expression of genes that were related to Fe uptake and transport as well as transcriptional regulators. In summary, our data reveal that low nitrate alleviated Fe deficiency through multiple pathway, which exhibits a new option for attenuating Fe deprivation by regulating the balance between nutrients.