Proteins function in cellular environments that are crowded with biomolecules, and in this reduced available space, their biophysical properties may differ from those observed in dilute solutions in vitro. Here, we investigated the effects of a synthetic macromolecular crowding agent, dextran 20, on the folded states of hyperthermophilic (S16T(herme)) and mesophilic (S161homologs of the ribosomal protein S16. As expected for an excluded-volume effect, the resistance of the mesophilic Meso, protein to heat-induced unfolding increased in the presence of dextran 20, and chemical denaturation experiments at different fixed temperatures showed the macromolecular crowding effect to be temperature-independent. Forster resonance energy transfer experiments show that intramolecular distances between an intrinsic Trp residue and BODIPY-labeled S16 Meso depend on the level of the crowding agent. The BODIPY group was attached at three specific positions in S16me, allowing measurements of three intraprotein distances. All S16meso variants exhibited a decrease in the average Trp-BODIPY distance at up to 100 mg/mL dextran 20, whereas the changes in distance became anisotropic (one distance increased, two distances decreased) at higher dextran concentrations. In contrast, the two 516-rhermo mutants did not show any changes in Trp-BODIPY distances upon increase of dextran 20 concentrations. It should be noted that the fluorescence quantum yields and lifetimes of BODIPY attached to the two S16 homologs decreased gradually in the presence of dextran 20. To investigate the origin of this decrease, we studied the BODIPY quantum yield in three protein variants in the presence of a tyrosine-labeled dextran. The experiments revealed distinct tyrosine quenching behaviors of BODIPY in the three variants, suggesting a dynamic local interaction between dextran and one particular S16 variant.