Soil water content (SWC) plays a crucial role in the production and transport of CO2 in soils. Classical approaches estimating the effects of SWC on soil respiration are incubation experiments, where soil structure is disturbed and transport processes are neglected. Nevertheless, such data govern the water reduction function of C turnover models. In our approach, the water reduction control parameters (WRCP) of a water reduction function were estimated from column experiments using inverse modeling. Therefore, we used the SOILCO2–RothC model in combination with multistep outflow (MSO) experiments. First, the effective hydraulic properties were estimated and then used in a second experiment to estimate the WRCP and rate constants of the resistant plant material (RPM) C pool. The results showed that the estimated hydraulic parameters can be used for the prediction of CO2 production and transport of a second MSO experiment only if the WRCP and the C turnover rate of the RPM pool of RothC will also be optimized. Optimizing only the WRCP matched the CO2 efflux fairly well but the WRCP at the highest matric potential, which determines the start of reduction, was too low at −1.61 cm and (water-filled pore space [WFPS] = 99.9%). Calibrating both WRCP and the RPM rate constant matched the efflux again fairly well and the results indicate a reduction of optimal CO2 production at water contents of 0.224 m3 m−3 or 53.3% WFPS. Also, the estimated RPM rate constant seems to be in a reasonable range at kRPM = 2.5791 × 10−7 cm−1.