Flaviviruses modify the lipid composition at viral replication sites and induce lipophagy to drive virion production. This study found that dengue virus (DENV), Zika virus (ZIKV) and West Nile virus (WNV) rely on the host lipid droplet-associated ancient ubiquitous protein 1 (AUP1) for lipophagy. The authors used proteomics to identify differentially ubiquitylated proteins in DENV-infected cells compared to uninfected cells and found that AUP1 became unconjugated during infection. AUP1 associates with DENV non-structural protein 4A (NS4A) and relocalizes from lipid droplets to autophagosomes. Virion production was significantly reduced in AUP1-depleted cells and in cells that expressed an AUP1 acyltransferase domain mutant. AUP1-knockout cells were also resistant to DENV, WNV and ZIKV infection. Ubiquitylation disrupted the AUP1–NS4A interaction and inhibited AUP1 acyltransferase activity, which is required for phospholipid biosynthesis. The authors conclude that flaviviruses exploit the acyltransferase activity of AUP1 for lipophagy, a process that is regulated by ubiquitylation.