Down syndrome (DS) is the most common chromosomal disorder and the leading genetic cause of intellectual disability in humans, which results from the triplication of chromosome 21. To search for biomarkers for the early detection and exploration of the disease mechanisms, here, we investigated the protein expression signature of peripheral blood mononuclear cells (PBMCs) in DS children compared with healthy donors (HD) by using an in-depth label-free shotgun proteomics approach. Identified proteins are found associated with metabolic pathways, cellular trafficking, DNA structure, stress response, cytoskeleton network, and signaling pathways. The results showed that a well-defined number of dysregulated pathways retain a prominent role in mediating DS pathological features. Further, proteomics results are consistent with published study in DS and provide evidences that increased oxidative stress and the increased induction of stress related response, is a participant in DS pathology. In addition, the expression levels of some key proteins have been validated by Western blot analysis while protein carbonylation, as marker of protein oxidation, was investigated. The results of this study propose that PBMCs from DS children might be in an activated state where endoplasmic reticulum stress and increased production of radical species are one of the primary events contributing to multiple DS pathological features.