BACKGROUND Vector-borne infectious diseases are caused by pathogenic microorganisms transmitted mainly by blood-sucking arthropod vectors. In laboratories, the handling of insects carrying human pathogens requires extra caution because of safety concerns over their escape risk. Based on standard insect containment practices, there have been cases where costly enhancements were required to definitely protect laboratory workers and neighbors from potential infection through mosquito bites. Here, we developed a mosquito rearing method that provides a reliable and cost-effective means to securely contain pathogen-infected females of the yellow fever mosquito Aedes aegypti. RESULTS To debilitate the motility of A. aegypti females, mosquitoes were rendered completely flightless by ablation of either wing. The "single-winged" mosquitoes exhibited a severe defect in flying ability and were incubated in a container with inside surfaces covered with a net stretched to approximately 1-mm mesh, which helped the mosquitoes hold on and climb up the wall. In this container, flightless females consistently showed similar blood feeding and egg laying activities to intact females. Eighty-five percent of the flightless mosquitoes survived at 1 week after wing ablation, ensuring feasibility of the use of these mosquitoes for studying pathogen dynamics. CONCLUSIONS This mosquito rearing method, with a detailed protocol, is presented here and can be readily implemented as a highly secure insectary for vectors carrying human pathogens. For researchers in an environment where highly strict containment practices are mandatory, this method could offer appropriate opportunities to perform research on pathogen-mosquito interactions in vivo.