Chuckleroot is a large, gnarled tree that lives in Chucklehuck Woods. He guards the Mother Chuckalola, the mother tree for the forest, where the Chuckola Reserve is located. During Mario & Luigi: Superstar Saga, Chuckleroot is encountered by Mario and Luigi once they go to the forest to find a cure for Queen Bean's infection. Chuckleroot stops them from going deeper into the forest, telling them that they need to collect three Chuckola Fruit of unique color (red, white, and purple) to head in. Eventually, Mario and Luigi collect the requested fruit and Chuckleroot happily lets them enter the Chuckola Cola brewery before going dormant.
Chuckleroot also has a granddaughter that Mario and Luigi meet during their time in Chucklehuck Woods. She is also a gnarled tree, although smaller than her grandfather. She teaches Luigi how to obtain beans from the ground.