Tsetse flies are insects in the family Glossinidae occurring in Africa which are blood-sucking pests of humans, domestic animals, and wildlife. They are known for disease transmission to humans (Human African Trypanosomiasis, HAT) and domestic and wild animals (Nagana). This chapter explores the biology and disease ecology of tsetse flies, particularly HAT. Lastly, diagnosis, laboratory findings, treatment and prevention of HAT and its vectors are presented and discussed.