Side-chain oxysterols are enzymatically generated oxidation products of cholesterol that serve a central role in mediating cholesterol homeostasis. Recent work has shown that side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), alter membrane structure in very different ways from cholesterol, suggesting a possible mechanism for how these oxysterols regulate cholesterol homeostasis. Here we extend our previous work by using molecular-dynamics simulations of 25-HC and cholesterol mixtures in 1-palmitoyl-2-oleoyl- phosphatidylcholine bilayers to examine the combined effects of 25-HC and cholesterol in the same bilayer. 25-HC causes larger changes in membrane structure when added to cholesterol-containing membranes than when added to cholesterol-free membranes. We also find that the presence of 25-HC changes the position, orientation, and solvent accessibility of cholesterol, shifting it into the water interface and thus increasing its availability to external acceptors. This is consistent with experimental results showing that oxysterols can trigger cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. These effects provide a potential mechanism for 25-HC-mediated regulation of cholesterol trafficking and homeostasis through modulation of cholesterol availability.