Rare earth elements, especially terbium (Tb), are high-valence heavy metal elements that accumulate in the environment, and they show toxic effects on plants. Signaling molecules regulate many physiological and biochemical processes in plants. How rare earth elements affect signaling molecules remains largely unknown. In the present study, the effects of Tb3+ on some extracellular and intracellular signaling molecules (gibberellic acid, abscisic acid, auxin, H2O2, and Ca2+) in horseradish leaves were investigated by using high-performance liquid chromatography, X-ray energy spectrometry, and transmission electron microscopy, and Tb3+ was sprayed on the surface of leaves. Tb3+ treatment decreased the auxin and gibberellic acid contents and increased the abscisic acid content. These changes in the contents of phytohormones (gibberellic acid, abscisic acid, and auxin) triggered excessive production of intracellular H2O2. Consequently, the increase in H2O2 content stimulated the influx of extracellular Ca2+ and the release of Ca2+ from Ca2+ stores, leading to Ca2+ overload and the resulting inhibition of physiological and biochemical processes. The effects outlined above were more evident with increasing the concentration of Tb3+ sprayed on horseradish leaves. Our data provide a possible underlying mechanism of Tb3+ action on plants.