HSP90 is an essential ATP-dependent molecular chaperone that facilitates the folding, the maturation and the activation of a wide range of proteins, called ‘clients'. Among the list of HSP90 clients there is transcription factors especially hormone receptors and kinases. Therefore HSP90 regulates numerous cellular processes such as: cell proliferation, cell survival and immunity. Our study aimed to understand the regulation of Hsp90 during skeletal muscle differentiation. We showed that the HSP90α mRNA levels remain constant during C2C12 myoblast differentiation, whereas the HSP90α protein levels were downregulated. Furthermore, we observed that HSP90β mRNA and protein levels remained constant during differentiation. Inhibition of the proteasomal and lysosomal degradation machinery allowed us to maintain some HSP90α protein during late stages of C2C12 cell differentiation. Taken together, these results suggest that the HSP90α protein is degraded by the proteasome and the lysosome during muscle differentiation. In the future, HSP90α ubiquitination needs to be confirmed in order to clarify the detailed mechanisms of degradation. Moreover, the regulation of key proteins for myogenesis by HSP90β should be investigated.