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Successful molecular cloning and functional characterization of a high-affinity urea permease ZmDUR3 provide convincing evidence of ZmDUR3 roles in root urea acquisition and internal urea-N-remobilization of maize plants. Urea occurs ubiquitously in both soils and plants. Being a major form of nitrogen fertilizer, large applications of urea assist cereals in approaching their genetic yield potential, but due to the low nitrogen-use efficiency of crops, this practice poses a severe threat to the environment through their hypertrophication. To date, except for paddy rice, little is known about the biological basis for urea movement in dryland crops. Here, we report the molecular and physiological characterization of a maize urea transporter, ZmDUR3. We show using gene prediction, PCR-based cloning and yeast complementation, that a functional full-length cDNA encoding a 731 amino acids-containing protein with putative 15 transmembrane α-helixes for ZmDUR3 was successfully cloned. Root-influx studies using 15N-urea demonstrated ZmDUR3 catalyzes urea transport with a K m at ~9 µM when expressed in the Arabidopsis dur3-mutant. qPCR analysis revealed that ZmDUR3 mRNA in roots was significantly upregulated by nitrogen depletion and repressed by reprovision of nitrogen after nitrogen starvation, indicating that ZmDUR3 is a nitrogen-responsive gene and relevant to plant nitrogen nutrition. Moreover, detection of higher urea levels in senescent leaves and obvious occurrence of ZmDUR3 transcripts in phloem-cells of mature/aged leaves strongly implies a role for ZmDUR3 in urea vascular loading. Significantly, expression of ZmDUR3 complemented atdur3-mutant of Arabidopsis, improving plant growth on low urea and increasing urea acquisition. As it also targets to the plasma membrane, our data suggest that ZmDUR3 functions as an active urea permease playing physiological roles in effective urea uptake and nitrogen remobilization in maize.