Flaviviruses encode a conserved, membrane-associated nonstructural protein 1 (NS1) with replication and immune evasion functions. The current knowledge of secreted NS1 (sNS1) oligomers is based on several low-resolution structures, thus hindering the development of drugs and vaccines against flaviviruses. Here, we revealed that recombinant sNS1 from flaviviruses exists in a dynamic equilibrium of dimer-tetramer-hexamer states. Two DENV4 hexameric NS1 structures and several tetrameric NS1 structures from multiple flaviviruses were solved at atomic resolution by cryo-EM. The stacking of the tetrameric NS1 and hexameric NS1 is facilitated by the hydrophobic β-roll and connector domains. Additionally, a triacylglycerol molecule located within the central cavity may play a role in stabilizing the hexamer. Based on differentiated interactions between the dimeric NS1, two distinct hexamer models (head-to-head and side-to-side hexamer) and the step-by-step assembly mechanisms of NS1 dimer into hexamer were proposed. We believe that our study sheds light on the understanding of the NS1 oligomerization and contributes to NS1-based therapies.