Abstract IFN-stimulated gene (ISG) 15 is a ubiquitin-like protein induced after type I IFN stimulation. There is a dearth of in vivo models to study free unconjugated ISG15 function. We found that free ISG15 enhances the production of IFN-γ and IL-1β during murine infection with Toxoplasma gondii. In our model, ISG15 is induced in a type I IFN–dependent fashion and released into the serum. Increased ISG15 levels are dependent on an actively invading and replicating parasite. Two cysteine residues in the hinge domain are necessary determinants for ISG15 to induce increased cytokine levels during infection. Increased ISG15 is concurrent with an influx of IL-1β–producing CD8α+ dendritic cells to the site of infection. In this article, we present Toxoplasma infection as a novel in vivo murine model to study the immunomodulatory properties of free ISG15 and uniquely link it to IL-1β production by CD8α+ dendritic cells driven by two cysteines in the hinge region of the protein.