Small heat shock proteins (sHsps) are intracellular molecular chaperones that prevent the aggregation and precipitation of partially-folded and destabilized proteins. sHsps are comprised of an evolutionarily conserved region of 80-100 amino acids denoted the α-crystallin domain which is flanked by regions of variable sequence and length: the N-terminal domain and the C-terminal extension. Whilst the two domains are known to be involved in organization of the quaternary structure of sHsps and interaction with their target proteins, the role of the C-terminal extension is enigmatic. Despite the lack of sequence similarity, the C-terminal extension of mammalian sHsps is typically a short, polar segment which is unstructured and highly flexible and protrudes from the oligomeric structure. Both the polarity and flexibility of the C-terminal extension are important for the maintenance of sHsp solubility and the complex it makes with its target protein. In the present study, mutants of murine Hsp25 were prepared in which the glutamic acid residues in the C-terminal extension at positions 190, 199 and 204 were each replaced with alanine. The mutants were found to be structurally altered and functionally impaired. Whilst there were no significant differences in the environment of tryptophan residues in the N-terminal domain or overall secondary structure, an increase in exposed hydrophobicity was observed for the mutants compared with wild type Hsp25. The average molecular masses of the E199A and E204A mutants were comparable to the wild type protein, while the E190A mutant was marginally smaller. All mutants displayed markedly reduced thermostability and chaperone activity compared with wild type. It is concluded that each of the glutamic acid residues in the C-terminal extension is important for Hsp25 to act as an effective molecular chaperone.