The glutamine-binding protein GlnBP is part of an ATP-binding cassette transporter system in Escherichia coli and uses two well-characterized conformational states, an open ligand-free and a closed-liganded state, to facilitate active amino-acid uptake. Existing literature on its ligand-binding mechanism lacked sufficient evidence to univocally assign the kinetic type of binding mechanism for GlnBP: ligand binding prior to conformational change, that is an induced fit, or the conformational selection, in which the ligand binds the matching conformation from a pre-existing ensemble. Since such mechanistic questions are relevant for our fundamental understanding of how this and other biomacromolecules regulate cellular processes, we here revisit the question for GlnBP. We present a biochemical and biophysical analysis using a combination of calorimetry, single-molecule and surface-plasmon resonance spectroscopy, and molecular dynamics simulations. We found that both apo- and holo-GlnBP s