High-fidelity protein localization is essential to define the identities and functions of different organelles and to maintain cellular homeostasis. Accurate localization of nascent proteins requires specific protein targeting pathways as well as quality control (QC) mechanisms to remove mislocalized proteins. The endoplasmic reticulum (ER) is the first destination for approximately one-third of the eukaryotic proteome and a major site of protein biosynthesis and QC. In mammalian cells, trafficking from the ER provides nascent proteins access to the extracellular space and essentially every cellular membrane and organelle except for mitochondria and possibly peroxisomes. Here, we discuss the biosynthetic mechanisms that deliver nascent proteins to the ER and the QC mechanisms that interface with the ER to correct or degrade mislocalized proteins.