Reproductive isolation prevents members of different species from producing offspring and is a prerequisite for speciation. Failure of communication between female tissues of the pistil and paternal pollen tubes represent major hybridization barriers in flowering plants. Polymorphic maternal LURE peptides and one identified male receptor PRK6 were involved in pollen tube attraction. Here we report that knock out of the whole Arabidopsis thaliana LURE1 gene family did not affect fertility, indicating that AtLURE1/PRK6-mediated signaling is dispensable for successful fertilization within one Arabidopsis species. Instead, we report a novel role of AtLURE1s as conspecific pollen tube emergence accelerators that favor conspecific pollen over pollen from other species and thus promote reproductive isolation. We further identified maternal XIUQIU1-4 peptides that attract pollen tubes in a non-species-specific manner. Cooperation between at least two types of attractants promotes fertilization success and the recently-evolved peptides AtLURE1s represent a key mechanism for reproductive isolation.