The nuclear factor κB (NF-κB) pathway plays essential roles in innate and adaptive immunity, but little is known how NF-κB signaling is compartmentalized and spatiotemporally activated in the cytoplasm. Here, we show that the lipogenesis signal cascade Scap-SREBP1-S1P/S2P orchestrates the homeostasis and spatiotemporal activation of NF-κB. SREBP cleavage-activating protein (Scap) and sterol regulatory element-binding protein 1 (SREBP1) form a super complex with inhibitors of NF-κB α (IκBα) to associate NF-κB close to the endoplasmic reticulum (ER). Upon lipopolysaccharide (LPS) stimulation, Scap transports the complex to the Golgi apparatus, where SREBP1 is cleaved by site-1 protease (S1P)/S2P, liberating IκBα for IκB kinase (Ikk)-mediated phosphorylation and subsequent activation of NF-κB. Loss of Scap or inhibition of S1P or S2P diminishes, while SREBP1 deficiency augments, LPS-induced NF-κB activation and subsequent inflammatory responses. Our results reveal the Scap-SREBP1 complex as an additional cytoplasmic checkpoint for NF-κB homeostasis and unveil the Golgi apparatus as the optimal cellular platform for NF-κB activation, providing insights into the crosstalk between lipogenesis signaling and immunity. Display omitted •Scap and SREBP1 form a complex with IκBα and associate NF-κB close to the ER•Scap-mediated Golgi apparatus translocation primes the NF-κB complex for activation•S1P/S2P-mediated SREBP1 cleavage on the Golgi apparatus triggers NF-κB activation•Compartmentalized NF-κB is essential for inflammatory response in vitro and in vivo Fei et al. demonstrate that the lipogenesis signal cascade Scap-SREBP1-S1P/S2P orchestrates the homeostasis and spatiotemporal activation of NF-κB during inflammation.