Sac1 is a conserved phosphoinositide phosphatase, whose loss-of-function compromises cell and organism viability. Here, we employ acute auxin-inducible Sac1 degradation to identify its immediate downstream effectors in human cells. Most of Sac1 is degraded in ~1 h, paralleled by increased PI(4)P and decreased cholesterol in the trans-Golgi network (TGN) during the following hour, and superseded by Golgi fragmentation, impaired glycosylation, and selective degradation of TGN proteins by ~4 h. The TGN disintegration results from its acute deacidification caused by disassembly of the Golgi V-ATPase. Mechanistically, Sac1 mediated TGN membrane composition maintains an assembly-promoting conformation of the V(0)a2 subunit. Key phenotypes of acute Sac1 degradation are recapitulated in human differentiated trophoblasts, causing processing defects of chorionic gonadotropin, in line with loss-of-function intolerance of the human SACM1L gene. Collectively, our findings reveal that the assembly of the Golgi V-ATPase is controlled by the TGN membrane via Sac1 fuelled lipid exchange.