Twilight Trail is a large, eerie, spooky trail that connects Twilight Town to Creepy Steeple. It is found and used during the events of Paper Mario: The Thousand-Year Door. The Twilight Trail is a dark area full of bare, dead trees and dark green grass; the ground on which Mario must travel is purple in color. During Chapter 4 of the game, Mario must travel back and forth throughout these woods many times in order to find "The Letter 'P'" to guess Doopliss' name, and defeat him. In order to gain access to the Twilight Trail, Mario must enter a pipe (which Flurrie must unveil, using her unique ability) and head towards the dark forest. Flurrie must also blow away certain parts of the woods to enable Mario to continue forth. In these woods, many enemies can be found. It is also hard to avoid contact with these enemies, as the paths in which they travel are considerably narrow. Towards the end of the forest, a large rock must be pushed in the background by Mario in order for its larger version in the path to move.

In a yellow ? Block in the woods' first screen, a Thunder Rage can be retrieved. In the second screen, a red ? Block holds the Hammer Throw badge.