Human neutrophils are an essential component of the innate immune response. Although significant progress has been made toward understanding mechanisms of phagocytosis and microbicidal activity, a comprehensive analysis of proteins comprising neutrophil phagosomes has not been conducted. To that end, we used subcellular proteomics to identify proteins associated with human neutrophil phagosomes following receptor-mediated phagocytosis. Proteins (n = 411 spots) resolved from neutrophil phagosome fractions were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and/or liquid chromatography (LC)-MS/MS analysis. Those associated with phagocytic vacuoles originated from multiple subcellular compartments, including the cytosol, plasma membrane, specific- and azurophilic granules, and cytoskeleton. Unexpectedly, several enzymes typically associated with mitochondria were identified in phagosome fractions. Furthermore, proteins characteristic of the endoplasmic reticulum (ER), including 11 molecular chaperones, were resolved from phagosome preparations. Confocal microscopy confirmed that proteins representing these major subcellular compartments were enriched on phagosomes of intact neutrophils. Notably, calnexin and glucose-regulated protein 78 co-localized with gp91phox in human neutrophils, and were thus likely delivered to phagosomes by fusion of specific granules. We conclude that neutrophil phagosomes have heretofore unrecognized complexity and function, which includes potential for antigen processing events.