Autophagy has been reported to be involved in supporting antigen cross‐presentation by dendritic cells (DCs). We have shown that DCs have the ability to store antigen for a prolonged time in endo‐lysosomal compartments and thereby sustain MHCI antigen cross‐presentation to CD8+ T cells. In the current study, we investigated the role of autophagy in long‐term antigen presentation. We show that the autophagy machinery has a negative impact on storage of antigen in DCs. Atg5–/– DCs which are deficient in autophagy or DCs treated with common autophagy inhibitors showed enhanced antigen storage and antigen cross‐presentation. This augmented antigen cross‐presentation effect is independent of altered proteasome enzyme activity or MHCI surface expression on DCs. We visualized that the storage compartments are in close proximity to LC3 positive autophagosomes. Our results indicate that autophagosomes disrupt antigen storage in DCs and thereby regulate long‐term MHCI cross‐presentation.