ClpB/Hsp104 efficiently reactivates protein aggregates in cooperation with the DnaK/Hsp70 system. As a member of the AAA+ protein family (i.e. an expanded superfamily of ATPases associated with diverse cellular activities), ClpB forms a ring-shaped hexamer in an ATP-dependent manner. A protomer of ClpB consists of an N-terminal domain (NTD), an AAA+ module, a middle domain and another AAA+ module. In the crystal structures, the NTDs point to two different directions relative to other domains and are not visible in the single-particle cryo-electron microscopy reconstruction, suggesting that the NTD is highly mobile. In the present study, we generated mutants in which the NTD was anchored to other domain by disulfide cross-linking and compared several aspects of ClpB function between the reduced and oxidized mutants, using the wild-type and NTD-truncated ClpB (ClpBΔN) as references. In their oxidized form, the mutants and wild-type bind casein with a similar affinity, although the affinity of ClpBΔN for casein was significantly low. However, the extent of casein-induced stimulation of ATPase, the rate of substrate threading and the efficiency of protein disaggregation of these mutants were all lower than those of the wild-type but similar to those of ClpBΔN. These results indicate that the NTD supports the substrate binding of ClpB and that its conformational shift assists the threading and disaggregation of substrate proteins.