Cerebrospinal fluid (CSF) is a body fluid of choice for biomarker studies of brain disorders but remains relatively under-studied compared to other biological fluids such as plasma, partly due to the more invasive means of its sample collection. The present study establishes an in-depth CSF proteome through the analysis of a unique CSF sample from a pool of healthy donors. After immuno-affinity depletion, the CSF sample was fractionated using off-gel electrophoresis and analyzed with liquid chromatography tandem mass spectrometry (MS) using the latest generation of hybrid Orbitrap mass spectrometers. The shotgun proteomic analysis allowed the identification of 20689 peptides mapping on 3379 proteins. To the best of our knowledge, the obtained dataset constitutes the largest CSF proteome published so far. Among the CSF proteins identified, 34% correspond to genes defined as elevated in the brain by The Human Protein Atlas. The principal Alzheimer disease biomarkers (e.g., tau protein, amyloid-β, apolipoprotein E and neurogranin) were detected. Importantly, our dataset significantly contributes to the Chromosome-Centric Human Proteome Project (C-HPP), and 13 proteins considered as missing are proposed for validation in accordance with the HPP guidelines.