Cadmium (Cd2+) interferes with the uptake, transport and utilization of several macro- and micronutrients, which accounts, at least in part, for Cd2+ toxicity in plants. However, the mechanisms underlying Cd2+ interference of ionic homeostasis is not understood. Using biophysical techniques including membrane potential measurements, scanning ion-selective electrode technique for non-invasive ion flux assays and patch clamp, we monitored the effect of Cd2+ on calcium (Ca2+) and potassium (K+) transport in root hair cells of rice. Our results showed that K+ and Ca2+ contents in both roots and shoots were significantly reduced when treated with exogenous Cd2+. Further studies revealed that three cellular processes may be affected by Cd2+, leading to changes in ionic homeostasis. First, Cd2+-induced depolarization of the membrane potential was observed in root hair cells, attenuating the driving force for cation uptake. Second, the inward conductance of Ca2+ and K+ was partially blocked by Cd2+, decreasing uptake of K+ and Ca2+. Third, the outward K+ conductance was Cd2+-inducible, decreasing the net content of K+ in roots. These results provide direct evidence that Cd2+ impairs uptake of Ca2+ and K+, thereby disturbing ion homeostasis in plants.