CO2 as a byproduct of organic waste/wastewater fermentation has an important impact on the carboxylate chain elongation. In this study, a semi-continuous flow reactor was used to investigate the effects of CO2 loading rates (Low = 0.5 LCO2·L−1·d−1, Medium = 1.0 LCO2·L−1·d−1, High = 2.0 LCO2·L−1·d−1) on chain elongation system Ethanol and acetate were utilized as the electron donor and electron acceptor, respectively. The results demonstrate that low loading rate of CO2 has a positive effect on chain elongation. The maximum production of caproate and CH4 were observed at a low CO2 loading rate. Caproate production reached 1.88 g COD·L−1·d−1 with a selectivity of 62.55 %, while CH4 production reached 129.7 ml/d, representing 47.4 % of the total. Metagenomic analysis showed that low loading rate of CO2 favored the enrichment of Clostridium kluyveri, with its abundance being 3.8 times higher than at of high CO2 loading rate. Metatranscriptomic analysis revealed that high CO2 loading rate induced oxidative stress in microorganisms, as evidenced by increased expression of heat shock proteins and superoxide dismutase genes. Further investigation suggested that genes associated with the reverse β-oxidation pathway, CO2 uptake pathway and hydrogenotrophic methanogenesis pathway were reduced at high CO2 loading rate. These findings provide insight into the underlying mechanisms of how CO2 affects chain elongation, and it could be a crucial reason for the poor performance of chain elongation systems with high endogenous CO2 production.