In the mammalian host, Trypanosoma congolense cytoadheres, or sequesters, to the vascular endothelium. Although sequestration influences clinical outcome, disease severity and organ pathology, its determinants and mediators remain unknown. Challenges such as the variability of animal models, the only-recently developed tools to genetically manipulate the parasite, and the lack of physiologically-relevant in vitro models have hindered progress. Here, we engineered brain and cardiac 3D bovine endothelial microvessel models that mimic the bovine brain microvasculature and the bovine aorta, respectively. By perfusing these models with two T. congolense strains, we investigated the roles of flow for parasite sequestration and tropism for different endothelial beds. We discovered that sequestration is dependent on cyclic adenosine monophosphate (cAMP) signalling, closely linked to parasite proliferation, but not associated with parasite transmission to the tsetse fly vector. Finally, by comparing the expression profiles of sequestered and non-sequestered parasites collected from a rodent model, we showed gene expression changes in sequestered parasites, including of surface variant antigens. This work presents a physiologically-relevant platform to study trypanosome interactions with the vasculature and provides a deeper understanding of the molecular and biophysical mechanisms underlying T. congolense sequestration.