Dendritic cells (DC) can process exogenous proteins for presentation on MHC class I (MHC-I) in a vesicular pathway (VP) that is distinct from the classical, cytosolic MHC-I pathway. Here we investigate the sensitivity of this VP to Brefeldin A (BFA), a fungal metabolite which inhibits vesicular trafficking by preventing the activation of some ADP-ribosylation factor (ARF) proteins, in both resting and differently activated DC. The VP could be directly visualized in DC by the presence of OVA-derived H-2K^b/SIINFEKL complexes in a LAMP1 positive, but EEA negative, compartment in wt but not in cathepsin S^−/− mice as these are unable to process OVA into the SIINFEKL peptide in endolysosomes. BFA, which binds to specific ARF-GDP-sec7 sites, both in the Golgi and in endolysosomes, was found to bind to and inhibit the VP in resting DC. If the VP was selectively activated with an immunostimulatory CpG ODN, binding to endolysosomal TLR9 receptors, or by the mere mechanical disruption of clustered DC cells, BFA no longer had this effect. The activation of the VP with both CpG and cellular disruption was found to be dependent on the MyD88 adaptor protein. We conclude that vesicular MHC-I processing in DC occurs in ARF-regulated LAMP1 positive vesicles which, as a consequence of cellular activation, no longer can bind BFA and thus become resistant to the inhibitory effect of this drug.