Living cells must be able to respond to physiological and environmental fluctuations that threaten cell function and viability. A cellular event prone to disruption by a wide variety of internal and external perturbations is protein folding. To ensure protein folding can proceed under a range of conditions, the cell has evolved transcriptional, translational, and posttranslational signaling pathways to maintain folding homeostasis during cell stress. This review will focus on oxidative protein folding in the endoplasmic reticulum (ER) and will discuss the features of the main facilitator of biosynthetic disulfide bond formation, Ero1. Ero1 plays an essential role in setting the redox potential in the ER and regulation of Ero1 activity is central to maintain redox homeostasis and proper ER folding activity.