Single-chain immunotoxins are ideal tools to selectively kill infectious agents. In applying this technology to block transmission of malaria parasites in the mosquito vector, we have constructed a single-chain immunotoxin composed of a single-chain antibody fragment (scFv) directed to Pbs2l on the surface of Plasmodium berghei ookinetes linked to a lytic peptide, Shiva-1. The single-chain immunotoxin was expressed in Escherichia coli, and the protein was purified by a Ni-NTA column. The single-chain immunotoxin was initially shown to exhibit greater killing properties for P. berghei ookinetes in vitro compared with the scFv or synthetic Shiva-1 peptide alone. In an attempt to block malaria transmission by genetically engineered bacteria, recombinant E. coli harboring the single-chain immunotoxin gene were introduced into the mosquito midgut by membrane feeding. The number of infected mosquitoes and their oocyst densities were significantly reduced when the mosquitoes were subsequently allowed to feed on P. berghei-infected mice. These results indicate not only that a single-chain immunotoxin with enhanced parasiticidal activity could form a basis for the development of more effective malaria therapeutic agents, but also that introduction of genetically engineered bacteria into anopheline mosquitoes may offer a practical approach to the regulation of malaria transmission.