Colloids have been recognized as key vectors of pollutants in aqueous environment. Amongst them, those formed by iron (Fe) and organic matter (OM) are of major importance due to their ubiquity in the surface environment and strong affinity for metals. In the recent years, Fe stable isotopes have been increasingly used to elucidate the sources and biogeochemical cycling of Fe in Earth's surface environments. In this study, we aim to elucidate (i) the possible Fe isotopic signature resulting from the Fe/OM colloid formation and (ii) the mechanisms involved in the development of such isotopic signature. For this purpose, Fe-OM associations were synthesized through binding and titration experiments. Various pH levels were used in order to study the isotope behavior of Fe occurring as free species at pH 1, as Fe-OM complexes at pH 2 and as mixed Fe-oxyhydroxide/OM nanoaggregates or particles at pH 6.5. Organic matter-free, Fe-free and OM membrane-deposition experiments were also performed. These suspensions were (ultra)filtered at 0.2 µm, 30 kDa and 5 kDa to evidence the possible Fe isotope fractionation between fractions. This protocol allowed also testing the potential of (ultra)filtration techniques to generate isotope fractionation. The results provided evidence that abiotic Fe precipitation, (ultra)filtration techniques and OM deposition were not able to produce significant Fe isotope fractionation under the experimental conditions. However, at circum-neutral pH, the Fe-OM binding and titration experiments displayed a significant enrichment of heavy Fe isotopes in the < 30 kDa fractions relative to the total Fe pool δ56Fe = 0.35 ± 0.05‰ and 0.26 ± 0.05‰ (95% confidence interval, 2σ and relative to international standard IRMM-14), respectively. Mass balance and error propagation calculation showed Fe isotope fractionation in binding and titration experiments between the > 30 kDa and < 30 kDa fractions for -0.35 ± 0.05‰ and -0.27 ± 0.05‰, respectively. This Fe isotope fractionation could be due to the complexation of Fe by OM in the < 30 kDa fractions. At pH 2, the OM-free experiment, the < 30 kDa fraction showed Fe isotope ratio δ56Fe = 0.75 ± 0.03‰ with an enrichment in heavy Fe isotopes of δ56Fe’ = 0.14 ± 0.04‰ relative the total Fe pool (δ56Fe’ is δ56Fe value which was corrected by δ56Fe of total fraction). This enrichment in heavy Fe isotopes induced an isotopic fractionation factor of -0.87 ± 0.26‰ between the > 30 kDa and < 30 kDa fractions produced by the complexation between the heavy Fe isotopes and OH- ligands in the < 30 kDa fraction. Natural Fe-OM associations were further investigated through oxidation experiments of a reduced wetland soil solution. The oxidized soil solution was (ultra)filtered at 5 µm, 3 µm, 0.2 µm, 30 kDa and 5 kDa. The highest δ56Fe was obtained in the smallest size fraction, i.e. < 5 kDa fraction, yielding a negative isotopic fractionation Δ56Fe >5kDa - <5kDa = -0.23 ± 0.08‰ suggesting that Fe heavy isotopes are preferentially bound to small humic OM molecules in the form of Fe monomers or small clusters. This study highlights the importance of organic matter for metals’ isotopic systems.