Updated project metadata. Accumulated unfolded proteins in the endoplasmic reticulum (ER) will trigger the unfolded protein response (UPR) to increase protein-folding capacity. The ER proteostasis and UPR signaling should be precisely and timely regulated. In the project, by unbiased proteomics analysis, we identify that the serine 357 of protein disulfide isomerase (PDI) is rapidly phosphorylated by the secretory pathway kinase Fam20C under ER stress. Remarkably, phosphorylated Ser357 induces an open conformation of PDI and turns it from a ‘foldase’ to a ‘holdase’, which is critical for preventing protein misfolding in the ER.