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The enzyme carbonic anhydrase (CA), which catalyzes the interconversion of bicarbonate with carbon dioxide (CO₂) and water, has been hypothesized to play a role in C₃photosynthesis. We identified two tobacco stromal CAs, β-CA1 and β-CA5, and produced CRISPR/Cas9 mutants affecting their encoding genes. While single knockout linesΔβ-ca1andΔβ-ca5had no striking phenotypic differences compared to wild type (WT) plants,Δβ-ca1ca5leaves developed abnormally and exhibited large necrotic lesions even when supplied with sucrose. Leaf development ofΔβ-ca1ca5plants normalized at 9,000 ppm CO₂. Leaves ofΔβ-ca1ca5mutants and WT that had matured in high CO₂had identical CO₂fixation rates and photosystem II efficiency. Fatty acids, which are formed through reactions with bicarbonate substrates, exhibited abnormal profiles in the chloroplast CA-less mutant. EmergingΔβ-ca1ca5leaves produce reactive oxygen species in chloroplasts, perhaps due to lower nonphotochemical quenching efficiency compared to WT.Δβ-ca1ca5seedling germination and development is negatively affected at ambient CO₂. Transgenes expressing full-length β-CA1 and β-CA5 proteins complemented theΔβ-ca1ca5mutation but inactivated (ΔZn-βCA1) and cytoplasm-localized (Δ62-βCA1) forms of β-CA1 did not reverse the growth phenotype. Nevertheless, expression of the inactivated ΔZn-βCA1 protein was able to restore the hypersensitive response to tobacco mosaic virus, whileΔβ-ca1andΔβ-ca1ca5plants failed to show a hypersensitive response. We conclude that stromal CA plays a role in plant development, likely through providing bicarbonate for biosynthetic reactions, but stromal CA is not needed for maximal rates of photosynthesis in the C₃plant tobacco.