Determining the soil hydraulic properties is a prerequisite to physically model transient water flow and solute transport in the vadose zone. Estimating these properties by inverse modelling techniques has become more common within the last 2 decades. While these inverse approaches usually fit simulations to hydrometric data, we expanded the methodology by using independent information about the stable isotope composition of the soil pore water depth profile as a single or additional optimization target. To demonstrate the potential and limits of this approach, we compared the results of three inverse modelling strategies where the fitting targets were (a) pore water isotope concentrations, (b) a combination of pore water isotope concentrations and soil moisture time series, and (c) a two-step approach using first soil moisture data to determine water flow parameters and then the pore water stable isotope concentrations to estimate the solute transport parameters. The analyses were conducted at three study sites with different soil properties and vegetation. The transient unsaturated water flow was simulated by solving the Richards equation numerically with the finite-element code of HYDRUS-1D. The transport of deuterium was simulated with the advection-dispersion equation, and a modified version of HYDRUS was used, allowing deuterium loss during evaporation. The Mualem–van Genuchten and the longitudinal dispersivity parameters were determined for two major soil horizons at each site. The results show that approach (a), using only the pore water isotope content, cannot substitute hydrometric information to derive parameter sets that reflect the observed soil moisture dynamics but gives comparable results when the parameter space is constrained by pedotransfer functions. Approaches (b) and (c), using both the isotope profiles and the soil moisture time series, resulted in good simulation results with regard to the Kling–Gupta efficiency and good parameter identifiability. However, approach (b) has the advantage that it considers the isotope data not only for the solute transport parameters but also for water flow and root water uptake, and thus increases parameter realism. Approaches (b) and (c) both outcompeted simulations run with parameters derived from pedotransfer functions, which did not result in an acceptable representation of the soil moisture dynamics and pore water stable isotope composition. Overall, parameters based on this new approach that includes isotope data lead to similar model performances regarding the water balance and soil moisture dynamics and better parameter identifiability than the conventional inverse model approaches limited to hydrometric fitting targets. If only data from isotope profiles in combination with textural information is available, the results are still satisfactory. This method has the additional advantage that it will not only allow us to estimate water balance and response times but also site-specific time variant transit times or solute breakthrough within the soil profile.