Proteinaceous inclusions in nerve cells and glia are a defining neuropathological hallmark in a variety of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). Their occurrence may be related to malfunctions of the proteolytic degradation systems. In cultured oligodendrocytes, proteasomal inhibition leads to protein aggregate formation resembling coiled bodies, which are characteristic for PSP and CBD. Large protein aggregates are excluded from the proteasome and can only be degraded by autophagy, a lysosomal pathway. Autophagy is a highly selective process, which requires a variety of receptor proteins for ubiquitinated proteins, such as p62 and histone deacetylase 6 (HDAC6). HDAC6 is mainly localized in the cytoplasm, and alpha-tubulin is its major substrate. HDAC6 is considered as a sensor of proteasomal stress; it is involved in the autophagosomal pathway and can mediate the retrograde transport of ubiquitinated proteins along the microtubules. As we have shown recently, HDAC6 is present in oligodendrocytes and its inhibition leads to morphological alterations, microtubule bundling, modulation of acetylation, and phosphorylation of the microtubule-associated protein tau. The present study was undertaken to investigate whether HDAC6 is involved in protein aggregate formation in oligodendrocytes and whether its inhibition modifies the consequences of MG-132-induced inhibition of the ubiquitin proteasome system (UPS). The data show that HDAC6 and acetylated tau are recruited to protein aggregates after proteasomal inhibition. Pharmacological inhibition of HDAC6 by the selective inhibitor tubastatin A (TST) and its small hairpin RNA (shRNA)-mediated downregulation alters the assembly of MG-132-induced compact protein aggregates. After TST treatment, they appear more diffusely dispersed throughout the cytoplasm. This is not a protective means but promotes the onset of apoptotic cell death. Furthermore, the heat shock response is altered, and TST suppresses the MG-132-stimulated induction of HSP70. To test whether the alteration of protein aggregate formation is related to the influence of HDAC6 on the autophagic degradation system, an oligodendroglial cell line, i.e., OLN-93 cells stably expressing green fluorescent protein (GFP)-microtubule associated protein light chain 3 (LC3) and tau, was used. During autophagosome formation, endogenous LC3 is processed to LC3-I, which is then converted to LC3-II. An increase of LC3-II is used as a reliable marker for autophagosome formation and abundance. It is demonstrated that inhibition of HDAC6 leads to the accumulation of LC3-positive autophagosomal vacuoles and an increase in LC3-II immunoreactivity, but the autophagic flux is rather impaired. Hence, the inhibition or dysregulation of HDAC6 contributes to stress responses and pathological processes in oligodendrocytes.