Significance The small heat shock protein αB-crystallin functions as an archetypical and ubiquitous molecular chaperone. It is an integral part of the cellular proteostasis system and associated with human diseases such as Alzheimer's disease, myopathy, cataract, and multiple sclerosis. The molecular architecture of αB-crystallin follows an intriguing construction plan characterized by a dynamic oligomer equilibrium. Here, we exploited phosphorylation mimetics as a tool to switch the protein to an activated functional state by a shift in the conformational ensemble. Using cryo-EM and image processing, we defined the structures of the activated αB-crystallin ensemble. Biochemical analysis revealed that, on activation, the N-terminal regions gain flexibility and solvent accessibility. This allows enhancing the activity of αB-crystallin and promoting its cooperation with the Hsp70 system.