The important role of natural organic matter (NOM)–Fe colloids in influencing contaminant transport, and this role can be influenced by the formation, aggregation, and particle deposition dynamics of NOM-Fe colloids. In this work, NOM-Fe colloids at different C/Fe ratios were prepared by mixing different concentrations of humic acid (HA) with 10 mg/L Fe(II) under anoxic conditions. The colloids were characterized by an array of techniques and their aggregation and deposition behaviors were examined under both anoxic and oxic conditions. The colloids are composed of HA-Fe(II) at anoxic conditions, while they are made up of HA-Fe(III) at oxic conditions until the C/Fe molar ratio exceeds 1.6. For C/Fe molar ratios above 1.6, the aggregation and deposition kinetics of HA-Fe(II) colloids under anoxic conditions are slower than those of HA-Fe(III) colloids under oxic conditions. Further, the aggregation of HA-Fe colloids under both anoxic and oxic conditions decreases with increasing C/Fe molar ratio from 1.6 to 23.3. This study highlights the importance of the redox transformation of Fe(II) to Fe(III) and the C/Fe ratio for the formation and stability of NOM-Fe colloids that occur in subsurface environments with anoxic–oxic interfaces.