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Abstract Carbon isotope biosignatures preserved in the Precambrian geologic record are primarily interpreted to reflect ancient cyanobacterial carbon fixation catalyzed by Form I RuBisCO enzymes. The average range of isotopic biosignatures generally follows that produced by extant cyanobacteria. However, this observation is difficult to reconcile with several environmental (e.g., temperature, pH, and CO₂concentrations), molecular, and physiological factors that likely would have differed during the Precambrian and can produce fractionation variability in contemporary organisms that meets or exceeds that observed in the geologic record. To test a specific range of genetic and environmental factors that may impact ancient carbon isotope biosignatures, we engineered a mutant strain of the model cyanobacteriumSynechococcus elongatusPCC 7942 that overexpresses RuBisCO across varying atmospheric CO₂concentrations. We hypothesized that changes in RuBisCO expression would impact the net rates of intracellular CO₂fixation versus CO₂supply, and thus whole‐cell carbon isotope discrimination. In particular, we investigated the impacts of RuBisCO overexpression under changing CO₂concentrations on both carbon isotope biosignatures and cyanobacterial physiology, including cell growth and oxygen evolution rates. We found that an increased pool of active RuBisCO does not significantly affect the¹³C/¹²C isotopic discrimination (ε_p) at all tested CO₂concentrations, yielding ε_pof ≈ 23‰ for both wild‐type and mutant strains at elevated CO₂. We therefore suggest that expected variation in cyanobacterial RuBisCO expression patterns should not confound carbon isotope biosignature interpretation. A deeper understanding of environmental, evolutionary, and intracellular factors that impact cyanobacterial physiology and isotope discrimination is crucial for reconciling microbially driven carbon biosignatures with those preserved in the geologic record.