Side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), are key regulators of cholesterol homeostasis. New evidence suggests that the alteration of membrane structure by 25-HC contributes to its regulatory effects. We have examined the role of oxysterol membrane effects on cholesterol accessibility within the membrane using perfringolysin O (PFO), a cholesterol-dependent cytolysin that selectively binds accessible cholesterol, as a sensor of membrane cholesterol accessibility. We show that 25-HC increases cholesterol accessibility in a manner dependent on the membrane lipid composition. Structural analysis of molecular dynamics simulations reveals that increased cholesterol accessibility is associated with membrane thinning, and that the effects of 25-HC on cholesterol accessibility are driven by these changes in membrane thickness. Further, we find that the 25-HC antagonist LY295427 (agisterol) abrogates the membrane effects of 25-HC in a nonenantioselective manner, suggesting that agisterol antagonizes the cholesterol-homeostatic effects of 25-HC indirectly through its membrane interactions. These studies demonstrate that oxysterols regulate cholesterol accessibility, and thus the availability of cholesterol to be sensed and transported throughout the cell, by modulating the membrane environment. This work provides new insights into how alterations in membrane structure can be used to relay cholesterol regulatory signals.