Interactions among ions and their specific interactions with macromolecular solutes are known to play a central role in biomolecular stability. However, similar effects in the conformational stability of protein loops that play functional roles, such as binding ligands, proteins, and DNA/RNA molecules, remain relatively unexplored. A well-characterized enzyme that has such a functional loop is Escherichia coli dihydrofolate reductase (ecDHFR), whose so-called M20 loop has been observed in three ordered conformations in crystal structures. To explore how solution ionic strengths may affect the M20 loop conformation, we proposed a reaction coordinate that could quantitatively describe the loop conformation and used it to classify the loop conformations in representative ecDHFR x-ray structures crystallized in varying ionic strengths. The Protein Data Bank survey indicates that at ionic strengths (I) below the intracellular ion concentration-derived ionic strength in E. coli (I ≤ 0.237M), the ecDHFR M20 loop tends to adopt open/closed conformations, and rarely an occluded loop state, but when I is >0.237M, the loop tends to adopt closed/occluded conformations. Distance-dependent electrostatic potentials around the most mobile M20 loop region from molecular dynamics simulations of ecDHFR in equilibrated CaCl2 solutions of varying ionic strengths show that high ionic strengths (I = 0.75/1.5M) can preferentially stabilize the loop in closed/occluded conformations. These results nicely correlate with conformations derived from ecDHFR structures crystallized in varying ionic strengths. Altogether, our results suggest caution in linking M20 loop conformations derived from crystal structures solved at ionic strengths beyond that tolerated by E. coli to the ecDHFR function.