In Europe, tick-borne encephalitis virus (TBEV) causes severe neurological disease in humans. As an obligate intracellular organism, TBEV relies on manipulating host metabolic processes and evading antiviral defenses for its survival. This is accomplished largely through binary interactions between specific viral proteins and host proteins. These protein-protein interactions (PPIs) constitute molecular determinants of critical pathobiologic traits of viruses, including host range, zoonotic potential and virulence, and making them realistic targets for antiviral therapies. To better understand the pathobiology of TBEV in humans, we mapped the network of PPIs between TBEV and its human host using interaction proteomics. We performed a high-throughput screen for virus-host PPIs by means of yeast two-hybrid methodology, utilizing the complete set of TBEV open reading frames and Homo sapiens cDNA libraries. This screen revealed a large number of human proteins that directly interact with TBEV viral proteins, many of which had not previously been associated with viral infections. We further investigated the functional significance of these host interactors in viral infection as viral dependency or restriction factors using RNA interference and bioinformatics analysis, identifying approximately 40% of the human proteins as having a significant impact on TBEV replication. These proteins are involved in diverse biological processes, some of which are expected to play a role in viral infection. Ongoing research is now focused on understanding the mechanisms of action of some of the most influential factors in the TBEV viral cycle. More broadly, this work aims to illuminate how tick-borne flaviviruses manipulate cellular processes to cause disease and to identify viral vulnerabilities that could be targeted therapeutically.