Nitrogen (N) speciation could influence the efficiency of Cd accumulation by plants grown in Cd-contaminated areas. However, limited information has been reported on how the application of N speciation to rice affects the Cd accumulation. Here, we investigated the physiological and genetic mechanisms involved in Cd uptake by roots, xylem translocation and subsequent Cd accumulation in rice (Oryza sativa L.) affected by different NO3−/NH4+ ratios under low-and-high Cd stress. Results showed that both N speciation and Cd stress affected plant growth, with high NH4+-N ratios treatments having higher tissue biomass, and high Cd treatment reducing tissue biomass. The Cd concentrations in shoots and roots were reduced with the increasing NH4+-N ratios (except for the full NO3−-N treatment), but did not affect the Cd translocation factor (TF). The total Cd accumulation of the whole plant were also reduced with the high NH4+-N ratios (NO3−/NH4+, 1:2 and 0:1) on the low Cd treatment due to the decline both in the Cd accumulation of shoots and roots. A scanning ion-selective electrode technique (SIET) showed that the net Cd2+ influxes at the root hair zone were inhibited with the increasing NH4+-N ratios in absence or pretreatment with different NO3−/NH4+ ratios. Additionally, the Cd concentrations in xylem sap were also displayed a decline trend with the increasing NH4+-N ratios under two dose Cd treatments. Furthermore, the gene expression related to Cd uptake (OsIRT1 and OsNRAPM5) and transport (OsHMA2) in roots showed the familiar tendencies with those of Cd uptake and transport at the physiological level treated with different NO3−/NH4+ ratios under two dose Cd treatments. It is concluded that increasing ammonium nutrition contributes to the inhibition of Cd uptake, xylem transport and subsequent accumulation in rice.