Plant root development depends on signaling pathways responding to external and internal signals. In this study we demonstrate the involvement of the Lotus japonicus LjNPF4.6 gene in the ABA and nitrate root responding pathways. LjNPF4.6 expression in roots is induced by external application of both nitrate and ABA. LjNPF4.6 promoter activity is spatially localized in epidermal cell layer and vascular bundle structures with the latter pattern being controlled by externally applied ABA. LjNPF4.6 cRNA injection achieves both nitrate and ABA uptake in Xenopus laevis oocytes and the analyses of L. japonicus knock-out insertion mutants confirmed the role played by LjNPF4.6 in root nitrate uptake. The phenotypic characterization of the Ljnpf4.6 plants indicates the role played by LjNPF4.6 in the root program development in response to exogenously applied nitrate and ABA. Based on the presented data, the mode of action of this transporter is discussed.