<jats:title>Abstract</jats:title> <jats:p>Pre-zygotic interspecies incompatibility in angiosperms is an important mechanism to prevent unfavorable hybrids between species. We used a genome-wide association study to identify Stigmatic privacy 2 (SPRI2), a transcription factor with a zinc-finger domain, which regulates interspecies barriers in Arabidopsis thaliana. CRISPR/Cas9-mediated genome-editing analyses indicated that <jats:italic>SPRI2</jats:italic> and its <jats:italic>SPRI2-like</jats:italic> paralog are required for rejection of the male pollen grains of other species in the female pistils. We also show that SPRI2 and SPRI2-like express interspecific incompatibility by directly regulating the mRNA transcription of two xylan O-acetyltransferases (TBL45 and TBL40) related to cell wall modification. In addition, they control the mRNA accumulation level of <jats:italic>SPRI1</jats:italic>, a key factor required for interspecific pollen rejection found in our previous study. SPRI2 is localized as condensed structures in the nucleus formed via liquid-liquid phase separation (LLPS). A prion-like sequence found in the N-terminal region of SPRI2 is responsible for the formation of the condensates. Our discovery of the LLPS-regulated SPRI2 has revealed the connections between transcriptional regulation, cell wall modification, and <jats:italic>SPRI1</jats:italic> in interspecific pollen rejection.</jats:p>