The hetero-oligomeric chaperonin of eukarya, TRiC, is required to fold the cytoskeletal protein actin. The simpler bacterial chaperonin system, GroEL/GroES, is unable to mediate actin folding. Here, we use spectroscopic and structural techniques to determine how TRiC promotes the conformational progression of actin to the native state. We find that actin fails to fold spontaneously even in the absence of aggregation but populates a kinetically trapped, conformationally dynamic state. Binding of this frustrated intermediate to TRiC specifies an extended topology of actin with native-like secondary structure. In contrast, GroEL stabilizes bound actin in an unfolded state. ATP binding to TRiC effects an asymmetric conformational change in the chaperonin ring. This step induces the partial release of actin, priming it for folding upon complete release into the chaperonin cavity, mediated by ATP hydrolysis. Our results reveal how the unique features of TRiC direct the folding pathway of an obligate eukaryotic substrate. Display omitted •Actin fails to fold spontaneously, strictly requiring TRiC chaperonin for folding•Actin binding to TRiC specifies a unique topology for productive folding•ATP binding induces an asymmetric TRiC intermediate and selective actin release•Stepwise folding on and inside TRiC allows actin to access the native state Single-molecule studies explain why the eukaryotic chaperone complex TRiC, but not the simpler bacterial chaperonin system, is able to induce the proper folding of actin.