Although genetically related, the tick-borne orthoflaviviruses tick-borne encephalitis (TBEV) and louping ill (LIV) show distinct pathogenicity across their host species. In sheep, LIV is known to cause fatal encephalitis, while TBEV often remains asymptomatic, whereas in humans, both viruses can induce severe neurological disease. The molecular mechanisms of this species-dependent pathogenesis remain largely unknown. In this study, we explored the role of endothelial cells in orthoflavivirus pathogenesis. We established physiologically relevant perfused 3D vessel-on-a-chip (VoC) models representing the sheep (shVoC) and human (hVoC) vasculature, using primary endothelial cells. Next, we assessed infectivity and replication kinetics of TBEV and LIV in these models. Furthermore, using transcriptomic analysis of infected cells, we aimed to identify differentially expressed genes (DEGs) between virus infections within each host species. Consistent with field observations, results show that while LIV exhibits similar growth kinetics in VoCs of both species, TBEV growth is significantly reduced in shVoCs compared to hVoCs. Interestingly, for both viruses, transcriptome analysis revealed a minimal number of DEGs in hVoCs. On the other hand, while <50 DEGs were found in TBEV-infected shVoCs, >3500 DEGs were found in LIV-infected shVoCs. Upregulated genes were associated with cytokine signaling and interferon-mediated immune responses (e.g., CCL5, IL-8, ISG15, MX2), as well as vascular activation and leukocyte adhesion (e.g., ICAM-1, VEGF, E-selectin). In addition to providing an in-depth understanding of orthoflavivirus pathogenesis, the developed VoCs can serve as tools for quickly assessing the virulence of newly arising viruses and contribute to the 3Rs in animal research.