Macrophages are immune cells that function in the clearance of infectious particles. This process involves the engulfment of microbes into phagosomes where these particles are lysed and degraded. In the current study, we used a large scale quantitative proteomics approach to analyze the changes in protein abundance induced on phagosomes by interferon-gamma (IFN-gamma), an inflammatory cytokine that activates macrophages. Our analysis identified 167 IFN-gamma-modulated proteins on phagosomes of which more than 90% were up-regulated. The list of phagosomal proteins regulated by IFN-gamma includes proteins expected to alter phagosome maturation, enhance microbe degradation, trigger the macrophage immune response, and promote antigen loading on major histocompatibility complex (MHC) class I molecules. A dynamic analysis of IFN-gamma-sensitive proteins by Western blot indicated that newly formed phagosomes display a delayed proteolytic activity coupled to an increased recruitment of the MHC class I peptide-loading complex. These phagosomal conditions may favor antigen presentation by MHC class I molecules on IFN-gamma-activated macrophages.