RsAFP2 (Raphanus sativus antifungal peptide 2), an antifungal plant defensin isolated from seed of R. sativus, interacts with glucosylceramides (GlcCer) in membranes of susceptible yeast and fungi and induces membrane permeabilization and fungal cell death. However, using carboxyfluorescein-containing small unilamellar vesicles containing purified GlcCer, we could not observe permeabilization as a consequence of insertion of RsAFP2 in such vesicles. Therefore, we focused on a putative RsAFP2-induced signaling cascade downstream of RsAFP2-binding to GlcCer in fungal membranes. We show that RsAFP2 induces reactive oxygen species (ROS) in Candida albicans wild type in a dose-dependent manner, but not at all in an RsAFP2-resistant DeltagcsC. albicans mutant that lacks the RsAFP2-binding site in its membranes. These findings indicate that upstream binding of RsAFP2 to GlcCer is needed for ROS production leading to yeast cell death. Moreover, the antioxidant ascorbic acid blocks RsAFP2-induced ROS generation, as well as RsAFP2 antifungal activity. These data point to the presence of an intracellular plant defensin-induced signaling cascade, which involves ROS generation and leads to fungal cell growth arrest.