Studies of neuronal, endocrine and metabolic disorders would be facilitated by characterization of the hypothalamus proteome. Protein extracts prepared from 16 whole rat hypothalami were measured by data independent label-free nanoflow liquid chromatography mass spectrometry (nLC-MSE). Peptide features were detected, aligned and searched against a rat SwissProt database using ProteinLynx Global Server v.2.5. The final combined dataset comprised 21,455 peptides, corresponding to 622 unique proteins, each identified by a minimum of two distinct peptides. The majority of the proteins (69%) were cytosolic, and 16% were membrane proteins. Important proteins involved in neurological and synaptic function were identified including several members of RAB family and proteins involved in glutamate biosynthesis.