Significance Lipid metabolism is crucial to many (patho-)physiological processes, including inflammation. In particular, cholesterol biosynthesis has emerged as an exciting novel therapeutic target in numerous recent studies. Much is known about the early steps in cholesterol biosynthesis; however, the later steps have hitherto largely been neglected. Here, we investigated the druggability of distal cholesterol biosynthesis using a selective and potent chemical probe (SH42). This inhibition leads to the accumulation of the bioactive metabolite desmosterol, boosting the biosynthesis of polyunsaturated fatty acids (PUFA) and the downstream production of antiinflammatory mediators. Our report integrates distal cholesterol biosynthesis and its intermediate desmosterol with endogenous PUFA biosynthesis and resolution of inflammation, rendering this pathway attractive for further developments in the context of proresolving therapies.