Self-incompatibility (SI) is an intraspecific reproductive barrier widely present in angiosperms. The SI system with the broadest occurrence in angiosperms is based on an S-RNase linked to a cluster of multiple S-locus F-box (SLF) genes found in the Solanaceae, Plantaginaceae, Rosaceae, and Rutaceae. Recent studies reveal that non-self S-RNase is degraded by the SCF^SLF-mediated ubiquitin-proteasome system in a collaborative manner in Petunia, but how self-RNase functions largely remains mysterious. Here, we show that S-RNases form S-RNase condensates (SRCs) in the self-pollen tube cytoplasm through phase separation and the disruption of SRC formation breaks SI in self-incompatible Petunia hybrida. We further find that the pistil SI factors of a small asparagine-rich protein HT-B and thioredoxin h (Trxh) together with a reduced state of the pollen tube all promote the expansion of SRCs, which then sequester several actin binding proteins, including the actin polymerization factor PhABRACL, whose actin polymerization activity is reduced by S-RNase in vitro. Meanwhile, we find that S-RNase variants lacking condensation ability fail to recruit PhABRACL and are unable to induce actin foci formation required for the pollen tube growth inhibition. Taken together, our results demonstrate that phase separation of S-RNase promotes SI response in P. hybrida, revealing a new mode of S-RNase action.