Under native conditions, proteins can undergo transient partial unfolding, which may cause proteins to misfold or aggregate. A change in sequence connectivity by circular permutation may affect the energetics of transient partial unfolding in proteins without altering the threedimensional structures. Using Escherichia coli dihydrofolate reductase (DHFR) as a model system, we investigated how circular permutation affects transient partial unfolding in proteins. We constructed three circular permutants, CP18, CP37, and CP87, with the new N-termini at residue 18, 37, and 87, respectively, and probed transient partial unfolding by native-state proteolysis. The new termini in CP18, CP37, and CP87 are within, near, and distal to the Met20 loop, which is known to be dynamic and also part of the region that undergoes transient unfolding in wild-type DHFR. The stabilities of both native and partially unfolded forms of CP18 are similar to those of wild-type DHFR, suggesting that the influence of introducing new termini in a dynamic region to the protein is minimal. CP37 has a significantly more accessible partially unfolded form than wild-type DHFR, demonstrating that introducing new termini near a dynamic region may promote transient partial unfolding. CP87 has significantly destabilized native and partially unfolded forms, confirming that modification of the folded region in a partially unfolded form destabilizes the partially unfolded form similar to the native form. Our findings provide valuable guidelines to control transient partial unfolding in designing circular permutants in proteins.