Physiological cholesterol metabolism implies the generation of a series of oxidized derivatives, whose oxysterols are by far the most investigated ones for their potential multifaceted involvement in human pathophysiology. In this regard, noteworthy is the broad antiviral activity displayed by defined side chain oxysterols, in particular 25-hydroxycholesterol (25HC) and 27-hydroxycholesterol (27HC). Although their antiviral mechanism(s) may vary depending on virus/host interaction, these oxysterols share the common feature to hamper viral replication by interacting with cellular proteins. Here reported is the first analysis of the modulation of a cell proteome by these two oxysterols, that, besides yielding additional clues about their potential involvement in the regulation of sterol metabolism, provides novelinsights about the mechanism underlying the inhibition of virus entry and trafficking within infected cells. We show here that both 25HC and 27HC can down-regulate the junction adhesion molecule-A (JAM-A) and the cation independent isoform of mannose-6-phosphate receptor (MPRci), two crucial molecules for the replication of all those viruses that exploit adhesion molecules and the endosomal pathway to enter and diffuse within target cells. Image 1 • 25HC and 27HC, two physiological products of enzymatic cholesterol oxidation, modulate the proteome of HeLa cells, standard model system. • Cell proteome analysis was afforded by coupling mass spectrometry to stable isotope labelling by amino acids in cell culture (SILAC) technology. • The large majority of proteins significantly modulated by both 25HC and 27HC is related to sterol synthesis and metabolism. • Down-regulation of JAM-A and MPRci likely contributes to the broad antiviral activity of 25HC and 27HC. [ABSTRACT FROM AUTHOR]