A review of current literature shows that considerable uncertainty exists concerning the role of the air–water interface (AWI) in colloid transport in unsaturated porous media. This study aimed to elucidate the mechanisms of colloid mobilization and retention at the AWI in a model dynamic system at the pore scale. The behavior of carboxylate-modified colloids during drainage and imbibition in a microfluidic channel was visualized with a confocal microscope. We found that dispersed hydrophilic colloids did not attach to the AWI under the investigated low ionic strength conditions, while colloids previously attached to the channel walls were mobilized by the contact line, retained at the AWI due to capillary forces, and thus were further transported. The study provides direct experimental evidence on the mechanisms by which colloid attachment to the AWI occurs and emphasizes the involvement of the moving contact line and AWI in colloid mobilization and transport in unsaturated porous media.