By means of a proteomic approach, we assessed the pathways involved in cerebellar neurodegeneration in a mouse model(Harlequin,Hq) of mitochondrial disorder. A differential proteomic profile study (iTRAQ) was performed in cerebellum homogenates of maleHqand wild-type (WT) mice 8 weeks after the onset of clear symptoms of ataxia in theHqmice (aged 5.2 ± 0.2 and 5.3 ± 0.1 months for WT andHq, respectively), followed by a biochemical validation of the most relevant changes. Additional groups of 2-, 3- and 6-month-old WT andHqmice were analyzed to assess the disease progression on the proteins altered in the proteomic study. The proteomic analysis showed that beyond the expected deregulation of oxidative phosphorylation, the cerebellum ofHqmice showed a marked astroglial activation together with alterations in Ca2+homeostasis and neurotransmission, with an up- and downregulation of GABAergic and glutamatergic neurotransmission, respectively, and the downregulation of cerebellar “long-term depression”, a synaptic plasticity phenomenon that is a major player in the error-driven learning that occurs in the cerebellar cortex. Our study provides novel insights into the mechanisms associated with cerebellar degeneration in theHqmouse model, including a complex deregulation of neuroinflammation, oxidative phosphorylation and glutamate, GABA and amino acids’ metabolism