The aim of this study is to analyze the influence of the soil moisture sampling depth in the parameterization of soil emission in microwave radiometry at L-band. The analysis is based on brightness temperature, soil moisture and temperature measurements acquired over a bare soil during the SMOSREX experiment. A more detailed profile of surface soil moisture was obtained with a soil heat and water flows mechanistic model. It was found that (1) the soil moisture sampling depth depends on soil moisture conditions, (2) the effective soil moisture sampling depth is shallower than provided by widely used field moisture sensors, and (3) the soil moisture sampling depth has an impact on the calibration of soil roughness model parameters. These conclusions are crucial for the calibration and validation of remote sensing data at L-band.