This study investigates the oligomeric states of secreted flavivirus nonstructural protein 1 (sNS1) using cryo-electron microscopy (cryo-EM). The authors found that recombinant sNS1 from various flaviviruses exists in a dynamic equilibrium of dimer, tetramer, and hexamer states. High-resolution structures of DENV4 NS1 dimer, tetramer, and hexamer were solved, revealing that the stacking of tetrameric and hexameric NS1 is facilitated by the hydrophobic β-roll and connector domains. A triacylglycerol molecule located within the central cavity may stabilize the hexamer. The study also proposed two models for the step-by-step assembly of NS1 dimer into hexamer: a head-to-head hexamer and a side-to-side hexamer. The findings contribute to the understanding of NS1 oligomerization and have implications for NS1-based therapies and vaccines.This study investigates the oligomeric states of secreted flavivirus nonstructural protein 1 (sNS1) using cryo-electron microscopy (cryo-EM). The authors found that recombinant sNS1 from various flaviviruses exists in a dynamic equilibrium of dimer, tetramer, and hexamer states. High-resolution structures of DENV4 NS1 dimer, tetramer, and hexamer were solved, revealing that the stacking of tetrameric and hexameric NS1 is facilitated by the hydrophobic β-roll and connector domains. A triacylglycerol molecule located within the central cavity may stabilize the hexamer. The study also proposed two models for the step-by-step assembly of NS1 dimer into hexamer: a head-to-head hexamer and a side-to-side hexamer. The findings contribute to the understanding of NS1 oligomerization and have implications for NS1-based therapies and vaccines.