Summary The ribosome‐inactivating protein BE 27 from sugar beet ( B eta vulgaris L .) leaves is an apoplastic protein induced by signalling compounds, such as hydrogen peroxide and salicylic acid, which has been reported to be involved in defence against viruses. Here, we report that, at a concentration much lower than that present in the apoplast, BE 27 displays antifungal activity against the green mould P enicillium digitatum , a necrotrophic fungus that colonizes wounds and grows in the inter‐ and intracellular spaces of the tissues of several edible plants. BE 27 is able to enter into the cytosol and kill fungal cells, thus arresting the growth of the fungus. The mechanism of action seems to involve ribosomal RNA ( rRNA ) N ‐glycosylase activity on the sarcin–ricin loop of the major r RNA which inactivates irreversibly the fungal ribosomes, thus inhibiting protein synthesis. We compared the C ‐terminus of the BE 27 structure with antifungal plant defensins and hypothesize that a structural motif composed of an α‐helix and a β‐hairpin, similar to the γ‐core motif of defensins, might contribute to the specific interaction with the fungal plasma membranes, allowing the protein to enter into the cell.