Genevestigator analysis has indicated heat shock induction of transcripts for NADPH-thioredoxin reductase, type C (NTRC) in the light. Here we show overexpression of NTRC in Arabidopsis (NTRC°(E)) resulting in enhanced tolerance to heat shock, whereas NTRC knockout mutant plants (ntrc1) exhibit a temperature sensitive phenotype. To investigate the underlying mechanism of this phenotype, we analyzed the protein's biochemical properties and protein structure. NTRC assembles into homopolymeric structures of varying complexity with functions as a disulfide reductase, a foldase chaperone, and as a holdase chaperone. The multiple functions of NTRC are closely correlated with protein structure. Complexes of higher molecular weight (HMW) showed stronger activity as a holdase chaperone, while low molecular weight (LMW) species exhibited weaker holdase chaperone activity but stronger disulfide reductase and foldase chaperone activities. Heat shock converted LMW proteins into HMW complexes. Mutations of the two active site Cys residues of NTRC into Ser (C217/454S-NTRC) led to a complete inactivation of its disulfide reductase and foldase chaperone functions, but conferred only a slight decrease in its holdase chaperone function. The overexpression of the mutated C217/454S-NTRC provided Arabidopsis with a similar degree of thermotolerance compared with that of NTRC°(E) plants. However, after prolonged incubation under heat shock, NTRC°(E) plants tolerated the stress to a higher degree than C217/454S-NTRC°(E) plants. The results suggest that the heat shock-mediated holdase chaperone function of NTRC is responsible for the increased thermotolerance of Arabidopsis and the activity is significantly supported by NADPH.