Cadmium (Cd) is nonessential and toxic for rice growth and humans. Rice is a major dietary source of Cd for Asian populations. Reducing Cd accumulation in rice grains is an important task. A number of transporters involved in the uptake or translocation of Cd have been identified in rice. Previous studies have shown that OsNRAMP5 is constitutively expressed in the roots and encodes a plasma membrane-localized protein. OsNRAMP5 is polarly localized at the distal side of both exodermis and endodermis cells. Knockout of OsNRAMP5 resulted in a significant reduction of Cd accumulation in rice roots and shoots. The aim of this study is to manipulate both the expression and tissue localization of OsNRAMP5 under the control of the rice OsActin1 promoter. We generated transgenic rice overexpressing OsNRAMP5, and tested the effect on Cd uptake and translocation. Overexpression of OsNRAMP5 in rice promoted Cd uptake into the roots but decreased Cd translocation from the roots to the shoots, and decreased Cd concentration in the shoots markedly. However, overexpression lines had no significant effect on manganese concentration of shoots, but increased Mn concentration of roots in hydroponic experiments when exposed to different Mn concentrations. A short-term Cd uptake experiment revealed that the K_m of overexpressing lines was more than two times higher than wild-type. Immunostaining showed that the overexpressed OsNRAMP5 proteins were localized in all cells of root tips and lateral root primordium without polarity. Cd imaging indicated the overexpression lines accumulated higher Cd concentration in the root tips by using synchrotron X-ray fluorescence microscopy (XFM). In addition, overexpressing lines showed much stronger intensity of Cd green fluorescens in the epithelial cells and vascular bundle of mature roots compared with the wild-type, in agreement with the results of the Cd concentration. When grown in two Cd-contaminated paddy soils, overexpression lines contained significantly lower Cd concentration in rice grains than the wild-type without disturbing accumulation of essential nutrients. Overexpression of OsNRAMP5 contributed to flux of Cd from the stele, restricting xylem loading of Cd consequently. Taken together, our study has shown that overexpression of OsNRAMP5 can reduce Cd accumulation in rice grains effectively.