Vanishing white matter (VWM) is a leukodystrophy caused by biallelic pathogenic variants in eukaryotic translation initiation factor 2B. To date, it remains unclear which factors contribute to VWM pathogenesis. Here, we investigated the basis of VWM pathogenesis using the 2b5 mouse model. We first mapped the temporal proteome in the cerebellum, corpus callosum, cortex, and brainstem of 2b5 and wild-type (WT) mice. Protein changes observed in 2b5 mice were then cross-referenced with published proteomic datasets from VWM patient brain tissue to define alterations relevant to the human disease. By comparing 2b5 mice with their region- and age-matched WT counterparts, we showed that the proteome in the cerebellum and cortex of 2b5 mice was already dysregulated prior to pathology development, whereas proteome changes in the corpus callosum only occurred after pathology onset. Remarkably, protein changes in the brainstem were transient, indicating that a compensatory mechanism might occur in this region. Importantly, 2b5 mouse brain proteome changes reflect features well-known in VWM. Comparison of the 2b5 mouse and VWM patient brain proteomes revealed shared changes. These could represent changes that contribute to the disease or even drive its progression in patients. Taken together, we show that the 2b5 mouse brain proteome is affected in a region- and time-dependent manner. We found that the 2b5 mouse model partly replicates the human disease at the protein level, providing a resource to study aspects of VWM pathogenesis by highlighting alterations from early to late disease stages, and those that possibly drive disease progression.