β/γ-Crystallins are the major structural proteins in mammalian lens. The N-terminal truncation of βB1-crystallin has been associated with the regulation of β-crystallin size distributions in human lens. Herein we studied the roles of βB1 N-terminal extension in protein structure and folding by constructing five N-terminal truncated forms. The truncations did not affect the secondary and tertiary structures of the main body as well as stability against denaturation. Truncations with more than 28 residues off the N-terminus promoted the dissociation of the dimeric βB1 into monomers in diluted solutions. Interestingly, the N-terminal extension facilitated βB1 to adopt the correct folding pathway, while truncated proteins were prone to undergo the misfolding/aggregation pathway during kinetic refolding. The N-terminal extension of βB1 acted as an intramolecular chaperone (IMC) to regulate the kinetic partitioning between folding and misfolding. The IMC function of the N-terminal extension was also critical to the correct refolding of β-crystallin heteromer and the action of the lens-specific molecular chaperone αA-crystallin. The cooperation between IMC and molecular chaperones produced a much stronger chaperoning effect than if they acted separately. To our knowledge, this is the first report showing the cooperation between IMC and molecular chaperones.