Fungi produce various defense proteins against antagonists including ribotoxins. These toxins cleave a single phosphodiester bond within the universally conserved sarcin-ricin loop of ribosomes and inhibit protein biosynthesis. Here, we report on the structure and function of ageritin, a previously reported ribotoxin from the edible mushroom The amino acid sequence of ageritin was derived from cDNA isolated from strain AAE-3 and lacks, according to prediction, a signal peptide for classical secretion, predicting a cytoplasmic localization of the protein. The calculated molecular weight of the protein is slightly larger than the one reported for native ageritin. The ageritin-encoding gene is highly induced during fruiting and toxicity assays with heterologously expressed in showed a strong toxicity against larvae, yet not against nematodes. The rRNase activity of recombinant ageritin was confirmed towards rabbit ribosomes. Mutagenesis studies revealed a correlation between and activities indicating that entomotoxicity is mediated by ribonucleolytic cleavage. The strong larvicidal activity of ageritin makes this protein a promising candidate for novel biopesticide development. Our results suggest a pronounced organismal specificity of a protein toxin with a very conserved intracellular molecular target. The molecular details of the toxin-target interaction will provide important insight into the mechanism of action of protein toxins and the ribosome. This insight might be exploited to develop novel bioinsecticides.