While it is recognized that preferential flow may increase the transport of colloids, less is known about the actual influence of preferential flow on colloid mobilization in situ. Changes in pore structure upon soil exposure to drying and rewetting may additionally affect colloid mobilization. Information about the pore structure and the active flow volume, as well as the changes in these properties, are therefore important when investigating colloid mobilization. We investigate the pore structure characteristics and the transport of tritium (3H2O) during steady unsaturated flow conditions. A total of 54 soil columns sampled along a natural clay gradient representing six clay contents (12, 18, 24, 28, 37, and 43% clay) were equilibrated to three different initial matric potentials (IMP), ψ = −2.5, −100, and −15500 hPa. Pore structure characteristics were deduced from water retention characteristics and measurements of air-filled porosity and air permeability. Tracer experiments were conducted at 1 mm h−1 and with a suction of 5 hPa. A mobile–immobile region model (MIM) and a three-region model (2MIM) with two mobile and one immobile region were used for describing the breakthrough curves (BTCs). The 2MIM model was able to fit the data well and predicted the existence of two mobile flow regions, most pronounced at higher clay content. The 12% clay soil exhibited matrix-dominated flow behavior, which is probably attributable to a large fraction of drained pores disconnecting the rapidly conducting flow system. Soils with ≥18% clay exhibited asymmetrical BTCs with early breakthrough and tailing and an increasing amount of immobile water, indicating preferential flow. Drying and rewetting, because of associated changes in the pore structure, significantly reduced the degree of preferential flow.