The Flavivirus non-structural glycoprotein NS1 is an unusual protein whose mechanistic function has remained elusive ever since it was first reported in 1970 as a viral antigen circulating in the sera of dengue infected patients. All Flavivirus NS1 proteins share a high degree of homology, encoding a 352 amino acid polypeptide that has a molecular weight of between 46 and 55 kDa depending on its glycosylation status. NS1 exists in multiple oligomeric forms and is found in different cellular locations. It is both cell membrane bound, in association with either virus induced intracellular vesicular compartments or on the cell surface and is also secreted as a soluble hexameric lipoparticle. The crystal structures of dengue virus and West Nile virus dimeric and hexameric forms of NS1 were recently reported, revealing the domains likely responsible for this membrane association but providing few clues to its functional role in the virus life cycle. Intracellular NS1 has been shown to co-localize with dsRNA and other components of the viral replication complex and is thought to play an essential cofactor role in virus replication. Although this makes NS1 an ideal target for inhibitor design, the precise nature of its cofactor function in viral replication has yet to be elucidated. A plethora of potential interacting partners have been identified, particularly for the secreted form of NS1, with many being implicated in immune evasion strategies. Secreted and cell surface associated NS1 are highly immunogenic and both the proteins themselves and the antibodies they elicit have been implicated in the seemingly contradictory roles of protection and pathogenesis in the infected host. NS1 has also been shown to be an important biomarker for early diagnosis of disease. An overview of these somewhat disparate areas of research will be presented.