African trypanosomes are flagellated protozoan parasites transmitted by the bite of tsetse flies and responsible for sleeping sickness in humans. Their complex development in the tsetse digestive tract requires several differentiation and migration steps that are thought to rely on trypanosome motility. We used a functional approach in vivo to demonstrate that motility impairment prevents trypanosomes from developing in their vector. Deletion of the outer dynein arm component DNAI1 results in strong motility defects but cells remain viable in culture. However, although these mutant trypanosomes could infect the tsetse fly midgut, they were neither able to reach the foregut nor able to differentiate into the next stage, thus failing to complete their parasite cycle. This is the first in vivo demonstration that trypanosome motility is essential for the accomplishment of the parasite cycle.