Protein transport into the endoplasmic reticulum (ER) is essential for all eukaryotic cells and evolutionary related to protein transport into and across the cytoplasmic membrane of eubacteria and archaea. It is based on amino-terminal signal peptides in the precursor polypeptides plus various transport components in cytosol plus ER and can occur either co- or posttranslationally. The two mechanisms merge at the heterotrimeric Sec61 complex in the ER membrane, which forms an aqueous polypeptide-conducting channel. Since the mammalian ER is also the main intracellular calcium storage organelle, the Sec61 complex is tightly regulated in its dynamics between the open and closed conformations by various ligands, such as precursor polypeptides at the cytosolic face, and the Hsp70-type molecular chaperone BiP at the ER lumenal face. Furthermore, BiP binding to the incoming precursor polypeptide contributes to unidirectionality and efficiency of transport. Recent insights into the structural dynamics of the Sec61 complex and related complexes in eubacteria and archaea have various mechanistic as well as functional implications.