The Escherichia coli MetNI-Q importer, an ATP-binding cassette (ABC) transporter, mediates the uptake of both L- and D- enantiomers of methionine. Original in vivo uptake studies show a strong preference for L-Met over D-Met, but the molecular basis of this selectivity is unclear. In this work, we systematically examine substrate discrimination by the MetNI transporter and MetQ substrate binding protein using an array of biophysical and biochemical techniques. Based on the kinetic and thermodynamic parameters of individual intermediates in the transport cycle, we uncover multiple steps in the transport cycle that confer substrate specificity. As in many other ABC importer systems, selectivity is applied at the level of binding to the substrate binding protein: MetQ dictates a 1,000-fold preference for L-Met over D-Met. However, beyond this initial level of selectivity, MetQ displays distinct binding preferences for the MetNI transporter depending on the substrate. We propose that the differences in binding affinities reflect the more favored release of L-Met into the permeation pathway when compared to D-Met. In support of this model, under saturating conditions, MetNI transports L-Met across the lipid bilayer at a faster rate than D-Met. Interestingly, the ATPase activity of the MetNI-Q complex is not modulated by the presence of substrate. Our studies reveal that the MetNI-Q system incorporates two separate steps in tuning methionine uptake to substrate chirality and availability. This method of discrimination ensures the import of the most biologically preferred substrate while also allowing for adaptability to more limiting nutrient conditions.