In recent years, different classes of proteins have been reported to promote toxic effects when ingested. Type-2 ribosome-inactivating proteins (RIPs) are a group of chimeric proteins built up of an A-chain with RNA N-glycosidase activity and a B-chain with lectin activity. These proteins are thought to play a role in plant protection. Sambucus nigra agglutinin I (SNA-I) is a type-2 RIP, isolated from the bark of elderberry (S. nigra L.). This study demonstrated the insecticidal potency of SNA-I on two Hemipteran insect species using two different methods. An artificial diet supplemented with different concentrations of the purified RIP reduced survival and fecundity of pea aphids Acyrthosiphon pisum. In addition, feeding of tobacco aphids, Myzus nicotianae, on leaves from transfected plants constitutively expressing SNA-I, resulted in a delayed development and reduced adult survival and also the fertility parameters of the surviving aphids were reduced, suggesting that a population of aphids would build up significantly slower on plants expressing SNA-I. Finally, a series of experiments with transgenic lines in which a mutant RIP was expressed, revealed that the carbohydrate-binding activity of SNA-I is necessary for its insecticidal activity. In a first set of mutants, the B-chain was mutated at one position (Asp231DeltaGlu), and in the second set both carbohydrate-binding sites were mutated (Asn48DeltaSer and Asp231DeltaGlu). Mutation of one carbohydrate-binding site strongly reduced the insecticidal activity of SNA-I, whereas mutation of both lectin sites (almost) completely abolished the SNA-I effect on tobacco aphids.