Iron-deficiency chlorosis is a common nutritional disorder in crops grown on alkaline or calcareous soils. Although the acclimation mechanism to iron deficiency has been investigated, the genetic regulation of iron acquisition is still unclear. Here, by comparing iron uptake process between an iron-poor-soil-tolerant citrus species Zhique (ZQ) and an iron-poor-soil sensitive citrus species trifoliate orange (TO), we discovered the enhanced root H+ efflux is crucial for the tolerance to iron deficiency in ZQ. The H+ efflux is mainly regulated by a plasma membrane localized H+-ATPase HA6, the expression of which is up-regulated in plants grown in soil with low iron content and significantly higher in the roots of ZQ than TO. Overexpression of the HA6 gene in Arabidopsis thaliana aha2 mutant defective in iron uptake recovered the wild type phenotype. In parallel, overexpression of the HA6 gene in TO significantly increased iron content of plants. Moreover, an iron deficiency-induced transcription factor MYB308 was revealed to bind the promoter and activate the expression of HA6 in ZQ by yeast one-hybrid, electrophoretic mobility shift and dual-luciferase assays. Overexpression of MYB308 in ZQ roots significantly increased the expression level of HA6 gene. However, MYB308 can not bind or activate the HA6 promoter in TO due to the sequence variation of corresponding MYB308 binding motif. Together, we propose that the MYB308 could activate HA6 to promote root H+ efflux and iron uptake, and the distinctive MYB308-HA6 transcriptional module may be, at least in part, responsible for the iron deficiency tolerance in citrus.