The game levels play similarly to those of Super Mario 3D Land, which combine the free-roaming gameplay of the Super Mario franchise's 3D games with the mechanics of its 2D side-scrollers, including a timer and a level-ending flagpole. Up to four concurrent players may control characters, who are Mario, Luigi, Princess Peach, Toad, and the unlockable Rosalina. Similar to their appearances in Super Mario Bros. 2, each of the characters possesses unique abilities and controls differently: Mario has balanced running speed and jump height; Luigi jumps higher and falls slower, but has slightly lower traction; Peach can jump and briefly float through the air, but runs slowly; Toad runs the fastest, but cannot jump as high and falls faster; Rosalina can use the spin attack move as seen in the Super Mario Galaxy games, but has the slowest running speed.  The player can select any of the five characters to use before beginning a level, and up to four players may explore the same level simultaneously, sharing from a pool of lives. Players are also able to pick up, carry, and throw each other. 
Levels are accessed through a world map, in which players can find hidden areas to earn more items or coins. Each level contains three collectable Green Stars, which are required to access certain levels, and each main level contains a hidden Stamp which was used in handwritten posts to the now defunct Miiverse online community. Players can also view messages left by other players, both on the world map and after clearing a level, and can download 'Mii Ghosts', which are live records of other players completing levels. Ghosts can be raced against, and may hold gift prizes such as coins or extra lives. Additional Green Stars can be earned in Captain Toad, Mystery House, and Challenge levels. 
Along with items returning from previous games, such as the Fire Flower, Super Leaf, Mega Mushroom, Propeller Box, and Boomerang Flower, several items are introduced. The Super Bell gives players a Cat Suit, allowing them to run faster, perform unique attacks and climb up walls to reach new areas. A variation of the Super Bell, the Lucky Bell, allows the player to temporarily transform their character into a lucky cat statue that awards coins under certain conditions. Double Cherries make a clone of the player, allowing for more effective attacks and a higher chance of survival; the more Double Cherries a player uses, the more clones appear. Players can wear Cannon Boxes which fire shots at enemies, Light Blocks which can defeat ghosts, and Goomba masks which let them blend in with enemy Goombas. Players are able to pick up various objects, such as bombs, baseballs, and Piranha Plants (which can be used to defeat enemies or solve puzzles), and can ride in a Skating Shoe or on an aquatic dinosaur named Plessie. 
The Wii U GamePad allows players to rub the touchscreen, blow into the microphone to reveal hidden blocks or items, hinder enemies, activate mechanisms, and use Off-TV Play. The game is compatible with the Wii Remote, Nunchuk, Classic Controller, and Wii U Pro Controller. An unlockable Luigi-themed version of Mario Bros., Luigi Bros., is accessed by clearing the game or by having save data from New Super Luigi U.