Abstract: Phosphoric acid is commonly used to react with carbonates to generate carbon dioxide for determining the carbon and oxygen isotopes of carbonates by using online reaction device, for example, GasBench coupled to an isotope ratio mass spectrometer (IRMS). During the measurement, oxygen isotope fractionation will arise due to various factors, which can result differences between the measured oxygen isotope values and the actual ones. In this study, a series of carbonate standard materials (international standard materials IAEA-603, NBS18, and national standard materials GBW04416 and GBW04405) and modern marine planktonic foraminiferal samples were determined to explore the contribution of acid fractionation on the oxygen isotope values under different conditions. The effects of acid fractionation on the oxygen isotope values for each standard material, and the underlying mechanism were investigated and discussed. The results showed that: 1) The acid fractionation effect is mainly caused by the exchange of oxygen isotopes between the sample CO₂ gas and the evaporated gas phase water in the headspace bottle (at high temperatures) or by the liquid water in the acid (at low temperatures); 2) The acid fractionation coefficients of oxygen isotope in carbonate reference materials with different characteristics show significant differences and are related to their oxygen isotope compositions; 3) The degree of deviation of oxygen isotopes is related to the factors such as reaction temperature, sample quantity, water content of phosphoric acid, and the difference between the oxygen isotope composition of liquid and gaseous water in the acid and the oxygen isotope composition of the samples; 4) The acid fractionation effect on oxygen isotopes is significantly correlated with signal intensity. It is recommended to effectively correct the oxygen isotope fractionation of acid by accurately weighing reference materials and samples, following the principle of peak intensity matching and adopting linear correction method for determination of oxygen isotopes in carbonates. Besides, a lower reaction temperature (25 ℃) generates slighter degree of oxygen isotope fractionation. This study provides a useful reference for the accurate determination of carbon and oxygen isotope composition in carbonates, and is of great significance for improving analysis accuracy.