After hearing about a strange inheritance dispute in the town of St. Mystere, Professor Layton and his young apprentice, Luke, travel there on behalf of Lady Dahlia, who has requested their presence to resolve the issue. However, it quickly becomes apparent that St. Mystere is quite strange... all the townsfolk seem to be obsessed with puzzles, and Baron Reinhold's fortune isn't the only secret that the town has hidden...

Professor Layton and the Curious Village is a puzzle game which uses the works of Akira Tago, author of the Japanese series of Head Gymnastics books as its basis. As both Layton and Luke, players explore the town of St. Mystere screen by screen. Each screen contains puzzles, either hidden in the scenery or by talking to characters. Upon finding a puzzle, the screen changes and the player is presented with the puzzle itself: the description of the puzzle on the top screen, and the puzzle (as well as the place to input the answer) on the bottom screen. Some puzzles require simple calculation, some have a trick answer, while others are riddles. The selection of puzzles is quite varied. If the player gets stuck, they can purchase a hint with 'Hint Coins'. However, these are limited - more can be found hidden in the game world, but there a set number of coins in the entire game.

Professor Layton has a visual style similar to that of a watercolor painting. While most of the game does not have much animation, key story sequences are told in fully animated and voice acted scenes. The game also used Nintendo Wi-Fi Connection to connect to the Internet and download additional puzzles for players to solve.