Listeria monocytogenes is a Gram-positive facultative anaerobic bacterium that is the etiological agent of listeriosis, a food-borne illness. In healthy individuals, listeriosis results in mild symptoms, but can be life-threatening for immunocompromised individuals such as the elderly or pregnant women in which case listeriosis can even lead to abortion. During infection with intracellular pathogens like L. monocytogenes, host cells upregulate a plethora of genes involved in combatting the infection. In infection with L. monocytogenes, one such gene is interferon stimulated gene 15 (ISG15), a ubiquitin-like protein, which has been shown to have antiviral activity. Previous work from our group demonstrated, through its effect on cytokines, that ISG15 is also antibacterial. However, we wanted to understand more about the protein's specific targets. Our group previously identified proteins targeted by ISG15 during L. monocytogenes infection using a combination of proteomic strategies. To confirm the modifications by ISG15 during L. monocytogenes infection, we infected mouse embryonic fibroblasts (MEFs) isolated from deconjugase (USP18) inactive mice, ISG15 knock-out mice, and wild type mice. Using infected cells, we found that ISG15 has a protective effect against L. monocytogenes infection in vitro. Additionally, since previous work has shown that approximately 80% of ISGylation targets are integral membrane proteins localized to the endoplasmic reticulum (ER), we focused specifically on morphological changes to the ER caused by ISG15 induction upon L. monocytogenes infection. Our data suggest that the absence of ISG15 may compromise both nuclear integrity and endoplasmic reticulum morphology. Although the role ISG15 plays upon infection with intracellular bacteria remains elusive, a better understanding of the mechanism of action of the small protein and its role during L. monocytogenes infection could lead to breakthroughs in the study of several human diseases due to the multitude of pathways in which ISG15 has been implicated.