To understand the mechanism underlying the ability of individual AdiC molecules to transport arginine and agmatine, we used a recently developed high-resolution single-molecule fluorescence-polarization microscopy method to investigate conformation-specific changes in the emission polarization of a bifunctional fluorophore attached to an AdiC molecule. With this capability, we resolved AdiC's four conformations characterized by distinct spatial orientations in the absence or presence of the two substrates, and furthermore, each conformation's two energetic states, totaling 24 states. From the lifetimes of individual states and state-to-state transition probabilities, we determined 60 rate constants characterizing the transitions and 4 KD values characterizing the interactions of AdiC's two sides with arginine and agmatine, quantitatively defining a 24-state model. This model satisfactorily predicts the observed Michaelis-Menten behaviors of AdiC. With the acquired temporal information and existing structural information, we illustrated how to build an experiment-based integrative 4D model to capture and exhibit the complex spatiotemporal mechanisms underlying facilitated transport of substrates. However, inconsistent with what is expected from the prevailing hypothesis that AdiC is a 1:1 exchanger, all observed conformations transitioned among themselves with or without the presence of substrates. To corroborate this unexpected finding, we performed radioactive flux assays and found that the results are also incompatible with the hypothesis. As a technical advance, we showed that a monofunctional and the standard bifunctional fluorophore labels report comparable spatial orientation information defined in a local frame of reference. Here, the successful determination of the complex conformation-kinetic mechanism of AdiC demonstrates the unprecedented resolving power of the present microscopy method.