Changes in the 12C/13C ratio (expressed as δ13C) of soil organic C (SOC) has been observed over long time scales and with depth in soil profiles. The changes are ascribed to the different reaction kinetics of 12C and 13C isotopes and the different isotopic composition of various SOC pool components. However, experimental verification of the subtle isotopic shifts associated with SOC turnover under field conditions is scarce. We determined δ13C and SOC in soil sampled during 1929–2009 in the Ap-horizon of five European long-term bare fallow experiments kept without C inputs for 27–80 years and covering a latitudinal range of 11°. The bare fallow soils lost 33–65 % of their initial SOC content and showed a mean annual δ13C increase of 0.008–0.024 ‰. The 13C enrichment could be related empirically to SOC losses by a Rayleigh distillation equation. A more complex mechanistic relationship was also examined. The overall estimate of the fractionation coefficient (ε) was −1.2 ± 0.3 ‰. This coefficient represents an important input to studies of long-term SOC dynamics in agricultural soils that are based on variations in 13C natural abundance. The variance of ε may be ascribed to site characteristics not disclosed in our study, but the very similar kinetics measured across our five experimental sites suggest that overall site-specific factors (including climate) had a marginal influence and that it may be possible to isolate a general mechanism causing the enrichment, although pre-fallow land use may have some impact on isotope abundance and fractionation.