beta-carbonic anhydrases (betaCA) accelerate the equilibrium formation between CO2 and carbonate. Two plant betaCA isoforms are targeted to the chloroplast and represent abundant proteins in the range of >1% of chloroplast protein. While their function in gas exchange and photosynthesis is well-characterized in carbon concentrating mechanisms of cyanobacteria and plants with C4-photosynthesis, their function in plants with C3-photosynthesis is less clear. The presence of conserved and surface-exposed cysteinyl residues in the betaCA-structure urged to the question whether betaCA is subject to redox regulation. Activity measurements revealed reductive activation of betaCA1, whereas oxidized betaCA1 was inactive. Mutation of cysteinyl residues decreased betaCA1 activity, in particular C280S, C167S, C230S, and C257S. High concentrations of dithiothreitol or low amounts of reduced thioredoxins (TRXs) activated oxidized betaCA1. TRX-y1 and TRX-y2 most efficiently activated betaCA1, followed by TRX-f1 and f2 and NADPH-dependent TRX reductase C (NTRC). High light irradiation did not enhance betaCA activity in wildtype Arabidopsis, but surprisingly in betaca1 knockout plants, indicating light-dependent regulation. The results assign a role of betaCA within the thiol redox regulatory network of the chloroplast.