The oxygen isotope composition of atmospheric CO2 (δ18Oac) can be used to disentangle ecosystem component CO2 fluxes, such as soil respiration and plant assimilation, because the δ18O composition of different water pools is transferred to CO2 during isotopic equilibration. The oxygen isotope exchange between CO2 and water in soils has been widely studied with theoretical models, but experimental data are scarce, albeit indispensable to characterization of the role of soils in determining δ18Oac. Here, we present a new methodology to monitor the δ18O of soil CO2 (δ18Osc) and of soil water (δ18Osw) in situ at varying soil water content. Infrared laser spectroscopy was combined with gas-permeable polypropylene (PP) tubing installed at different depths in a sand column. The permeable tubing did not lead to any isotopic fractionation and was suitable for combined δ18Osc and δ18Osw measurements. Soil water became gradually 18O enriched from the top of the sand over several days. Measured and δ18Osc simulated with the model MuSICA indicated incomplete CO2–H2O isotopic equilibrium. Irrigation of the sand column with tapwater resulted in a temporary reset of δ18Osw along the soil column, while δ18Osc was only influenced when the enzyme carbonic anhydrase was added to the irrigation water. Our study demonstrates that δ18Osc and δ18Osw can now be monitored in situ and online with high time resolution with minimum disturbance. With this new tool at hand, research into the oxygen isotope exchange between soil water and CO2 in natural soils has the potential to advance to a new stage and help to constrain the atmospheric CO2 budget.