In S-RNase-based self-incompatibility, S-RNase was previously thought to function as a selective RNase that inhibits pollen whose S-haplotype matches that in the pistil. In this study, we showed that S-RNase has a distinct effect on the regulation of Ca2+-permeable channel activity in the apical pollen tube in Pyrus pyrifolia. While non-self S-RNase has no effect, self S-RNase decreases the activity of Ca2+ channels and disrupts the Ca2+ gradient at the tip of the growing pollen tube during the gametophytic self-incompatibility (GSI) response. Extracellular Ca2+ influx was suppressed 5min after self S-RNase treatment, and self-pollen tube growth was reduced at 50min after self S-RNase treatment. In the self-incompatible response, the expression of Ca2+-related genes was inhibited before RNA degradation. Therefore, self S-RNase suppresses Ca2+ influx prior to arresting pollen tube growth via RNA degradation.