This study investigated in situ colloid mobilization and leaching from unsaturated, undisturbed soil columns to evaluate the processes controlling colloid mobilization in structured soils. 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 (wet), −100 (moderately wet), and −15500 hPa (dry) followed by 5 d of irrigation at 1 mm h−1 and applying a suction of 5 hPa at the lower boundary. The results revealed that (i) colloid leaching from the initially wet and moderately wet soils decreased with increasing clay content, (ii) colloid leaching from the initially dry soils was low and independent of clay content, and (iii) the leaching of total organic C (TOC) consisted mainly of dissolved organic C (DOC), and drying to −15500 hPa increased the leaching of C. In situ colloid mobilization and leaching was related to measurements of low-energy water-dispersible colloids (LE-WDC). Results indicate that in situ colloid mobilization from the initially wet and moderately wet 12% clay soils subjected to matrix-dominated flow behavior was controlled mainly by the time-dependent increase in colloid dispersion, while colloid mobilization from the initially dry soils was limited by the strong and persistent association created during the drying. In the more clayey soils, which were dominated by preferential flow, a lower displacement of high–ionic strength soil water with low–ionic strength rainwater may contribute to the inherently lower dispersibility in controlling colloid mobilization.