A growing number of field studies report isotopic offsets between stem water and its potential sources that prevent the unambiguous identification of plant water origin using water isotopes. We explored the causes of this isotopic offset by conducting a controlled experiment on the temperate tree species Fagus sylvatica. We measured delta H-2 and delta O-18 of soil and stem water from potted saplings growing on three soil substrates and subjected to two watering regimes. Regardless of substrate, soil and stem water delta H-2 were similar only near permanent wilting point. Under moister conditions, stem water delta H-2 was 11 +/- 3 parts per thousand more negative than soil water delta H-2, coherent with field studies. Under drier conditions, stem water delta H-2 became progressively more enriched than soil water delta H-2. Although stem water delta O-18 broadly reflected that of soil water, soil-stem delta H-2 and delta O-18 differences were correlated (r = 0.76) and increased with transpiration rates indicated by proxies. Soil-stem isotopic offsets are more likely to be caused by water isotope heterogeneities within the soil pore and stem tissues, which would be masked under drier conditions as a result of evaporative enrichment, than by fractionation under root water uptake. Our results challenge our current understanding of isotopic signals in the soil-plant continuum.