AbstractMembrane lipids play vital roles in small extracellular vesicle (sEV) biogenesis. However, the function of various lipids in the biogenesis of sEVs is still poorly understood. Phosphoinositolphosphates (PIPs), a group of the most critical lipids in vesicle transport, can undergo rapid conversion in response to a variety of cell signals, which in turn influence the generation of vesicles. Due to the challenge in detecting the low amount of PIP content in biological samples, the function of PIPs in sEVs has been insufficiently investigated. Here, we employed an LC‐MS/MS method to detect the levels of PIPs in sEVs. We revealed phosphatidylinositol‐4‐phosphate (PI4P) was the main PI‐monophosphate in macrophage‐derived sEVs. The release of sEVs was regulated in a time‐dependent manner and correlated with the PI4P level during the lipopolysaccharide (LPS) stimulation. In terms of mechanism, within 10 h of LPS treatment, the LPS‐induced production of type I interferon inhibited the expression of PIP‐5‐kinase‐1‐gamma, which increased the PI4P content on multivesicular bodies (MVBs) and recruited RAB10, member RAS oncogene family, to promote sEV generation. When LPS stimulation was extended to 24 h, the heat shock protein family A member 5 (HSPA5) expression level was elevated. PI4P interacted with HSPA5 on the Golgi or endoplasmic reticulum away from MVBs, which disrupted the continuous fast sEV release. In conclusion, the present study demonstrated an inducible sEV release model response to LPS treatment. The inducible release may be due to PI4P regulating the generation of intraluminal vesicles secreted as sEVs.