Spermidine (Spd) has been correlated with various physiological and developmental processes in plants, including pollen tube growth. In this work, we show that Spd induces an increase in the cytosolic Ca2+ concentration that accompanies pollen tube growth. Using the whole-cell patch clamp and outside-out single-channel patch clamp configurations, we show that exogenous Spd induces a hyperpolarization-activated Ca2+ current: the addition of Spd cannot induce the channel open probability increase in excised outside-out patches, indicating that the effect of Spd in the induction of Ca2+ currents is exerted via a second messenger. This messenger is hydrogen peroxide (H2O2), and is generated during Spd oxidation, a reaction mediated by polyamine oxidase (PAO). These reactive oxygen species trigger the opening of the hyperpolarization-activated Ca2+-permeable channels in pollen. To provide further evidence that PAO is in fact responsible for the effect of Spd on the Ca2+-permeable channels, two Arabidopsis mutants lacking expression of the peroxisomal-encoding AtPAO3 gene, were isolated and characterized. Pollen from these mutants was unable to induce the opening of the Ca2+-permeable channels in the presence of Spd, resulting in reduced pollen tube growth and seed number. However, a high Spd concentration triggers a Ca2+ influx beyond the optimal, which has a deleterious effect. These findings strongly suggest that the Spd-derived H2O2 signals Ca2+ influx, thereby regulating pollen tube growth.