Roads impact wildlife through changes in behavior, energetics, health, immune function, reproductive rates, and movement and migration. In recent years, there is increasing interest in understanding how roads affect physiological processes that, in turn, influence larger phenotypic and species-level impacts. Here, we discuss the role of glucocorticoids (colloquially termed “stress” hormones) in modulating interactions between wildlife and roads. Specifically, we discuss our current understanding of how roads affect wildlife via changes in glucocorticoids, different methodological approaches to quantify glucocorticoid physiology across taxa and contexts, and future directions for research. Uncovering the mechanistic underpinnings of what drives road-related impacts on wildlife is crucial for developing strategies to effectively mitigate them in an increasingly anthropogenic and interconnected world.