Multi-domain proteins, containing several structural units within a single polypeptide, constitute a large fraction of all proteomes. Co-translational folding is assumed to simplify the conformational search problem for large proteins, but the events leading to correctly folded, functional structures remain poorly characterized. Similarly, how the ribosome and molecular chaperones promote efficient folding remains obscure. Using optical tweezers, we have dissected early folding events of nascent elongation factor G, a multi-domain protein that requires chaperones for folding. The ribosome and the chaperone trigger factor reduce inter-domain misfolding, permitting folding of the N-terminal G-domain. Successful completion of this step is a crucial prerequisite for folding of the next domain. Unexpectedly, co-translational folding does not proceed unidirectionally; emerging unfolded polypeptide can denature an already-folded domain. Trigger factor, but not the ribosome, protects against denaturation. The chaperone thus serves a previously unappreciated function, helping multi-domain proteins overcome inherent challenges during co-translational folding. Display omitted •How the ribosome modulates nascent chain folding switches during elongation•Sequential domain-wise folding reduces misfolding•Co-translational folding can be reversed by an unexpected unfolding pathway•Protection of folded domains is an unanticipated chaperone function Liu et al. show that domain-wise folding of nascent proteins can be reversed by denaturing interactions with emerging polypeptide. The chaperone trigger factor blocks denaturation and, together with the ribosome, reduces misfolding. The chaperone thus serves a dual function in promoting efficient folding of multi-domain proteins.