Heat shock protein 60 (Hsp60) functions as a molecular chaperone, playing a vital role in maintaining protein homeostasis by regulating protein folding and preventing protein aggregation. Levels of heat shock proteins increase in response to oxidative stress and mitochondrial damage, both of which are symptoms commonly associated with various forms of epilepsy. Hsp60 levels are increased in the plasma of patients with temporal lobe epilepsy (TLE). It is crucial to understand what conformations and functions Hsp60 obtains within its folding cycle to determine Hsp60’s role in seizure response. First, we sought to visualize Hsp60’s folding mechanisms using advanced techniques, including nuclear magnetic resonance (NMR) spectroscopy, cryo-electron microscopy (cryo-EM), and X-ray crystallography. In contrast to its bacterial counterpart GroEL, which is always a stable tetradecamer, Hsp60 can adopt various states and the absence of intermediate structures remains a notable gap in our current understanding. Second, we determined the role of the folding cycle intermediates through functionality assays recording the folding of its natural substrates and ATPase activity. Surprisingly, Hsp60 is also active without its co-chaperone, Hsp10, although to a lesser extent. Understanding the complete folding cycle of Hsp60 will provide valuable insight for developing much-needed biomarkers and eventually drugs for temporal lobe epilepsy (TLE).