Down syndrome is a chromosomal alteration characterised by a complex symptomatology affecting several organs including the brain. It is the most frequent cause of intellectual disability of genetic origin and among the cognitive processes most affected are memory and language. While there have been advances in surgery and rehabilitation to improve physical symptoms, there are no pharmacological approaches to date for intellectual ones. The precise mechanisms by which the triplication of HSA21 genes leads to neuroanatomical and neurobehavioral phenotypes in individuals with DS are still poorly understood. In this work, we took a multi-omic approach and simultaneously analyze by RNA-seq and proteomics the expression signature of hippocampus and cortex of human postmortem DS and control brains to identify dysregulated expression networks in DS. Our data provided molecular information that consistently repeated at gene, transcript, and protein levels and identify new possible critical players in the pathophysiology of DS. We observed alteration of RNA splicing specifically altering the expression of genes involved in cytoskeleton and axonal dynamics in DS brains. Accordingly, we found alteration in axonal polarization in neurons from DS human induced-pluripotent-stem cells and mice. Thus, our study provides an integrated multi-layer expression database capable of identifying new potential targets to possibly aid design future clinical interventions in DS capable of enhancing existing cognitive abilities, improving learning and memory skills.