With a few exceptions, the gameplay in Star Fox 64 3D is very similar to that of the original N64 version. The player controls Fox's Arwing fighter jet using the Circle Pad to steer, the shoulder buttons to bank left and right, and the four right-hand buttons to fire lasers and bombs, boost, and brake. The directional pad allows the player to perform somersaults and U-Turns, which can also be performed with combinations of other controls, and to zoom in and out from the Arwing in "All-Range Mode". The player can also enable "Gyro Controls", using the 3DS's internal gyroscope sensor to control the Arwing. Character dialogue, messages, and control information are displayed on the bottom screen.
The single-player campaign features two modes: "64 Mode", which replicates the original game's objectives and difficulty, and "3DS Mode", in which difficulty and objectives have been tuned and balanced to account for the 3D perspective and gyro controls. A third "Expert" mode can be unlocked during gameplay, adding the difficulty of the original game's expert setting. Each mode separately tracks the player's scores and medals.