Quantification of clinically meaningful tibiofemoral motions requires a joint coordinate system (JCS) with motions free from kinematic crosstalk errors. The objectives were to use a JCS with literature-backed functional axes (FUNC) and a JCS recommended by the International Society of Biomechanics (ISB) to determine tibiofemoral kinematics of the native (i.e., healthy) knee, determine variability associated with each JCS, and determine whether the FUNC JCS significantly reduced kinematic crosstalk errors compared to the ISB JCS. Based on a kinematic model consisting of a three-cylindric joint chain, the FUNC JCS included functional flexion–extension (F–E) and internal-external (I–E) tibial rotation axes. In contrast, the ISB JCS included F–E and I–E axes defined using anatomic landmarks. Single-plane fluoroscopic images in 13 subjects performing a weighted deep knee bend were analyzed. Tibiofemoral kinematics using the FUNC JCS fell within the physiological range of motion in all six degrees-of-freedom. Internal tibial rotation averaged 13 deg for the FUNC JCS versus 10 deg for the ISB JCS and motions in the other four degrees-of-freedom (collectively termed off-axis motions) were minimal as expected based on biomechanical constraints. Off-axis motions for the ISB JCS were significantly greater; maximum valgus rotation was 4 deg and maximum anterior and distraction translations were 9 mm and 25 mm, respectively, which is not physiologic. Variabilities in off-axis motions were significantly greater with the ISB JCS (p < 0.0002). The FUNC JCS achieved clinically meaningful kinematics by significantly reducing kinematic crosstalk errors and is the more suitable coordinate system for quantifying tibiofemoral motions.