Dissolved organic matter (DOM) is a heterogeneous mixture of organic compounds that plays an important role in the movement of DOM-associated pollutants. In this study, transport and fractionation of DOM in soils was investigated in flow-through soil columns. Dissolved organic matter derived from spent mushroom substrate weathering was pumped through packed columns (2.5 cm × 10 cm) comprising a coarse-loamy subsoil (mixed, semiactive, mesic Typic Hapludult), and effluents were monitored for changes in the composition of DOM. Effluent DOM was characterized for UV absorbance, molecular weight, acidity, and hydrophilicity. Transport through the columns resulted in preferential retention of specific DOM constituents as indicated by comparison with a Br−tracer. During the transport process, effluent DOM exhibited decreasing values of E2/E3(from 10.3 to 6.2), acidity (from 20.8 to 13.1 mmolcg−1C), and hydrophilicity (39.0 to 28.4%), and increasing values of molar absorptivity (from 164 to 310 L mol−1C cm−1) and number and weight-averaged molecular weight (from 1770 to 3150 and 2450 to 4180 Da, respectively). These results indicate that DOM fractions with higher molecular weight, higher molar absorptivity, lower E2/E3ratio, lower acidity, and lower hydrophilicity were adsorbed preferentially by soil minerals, whereas the inverse fractions were transported preferentially. The adsorbed DOM could not be completely desorbed by DOM-free background solution, indicating a strongly bound fraction. Sorptive fractionation of DOM during transport likely affects the transport behavior of DOM-complexed constituents.