The dependence of the preferred orientation of polystyrene microcubes on surface hydrophobicity at the water/hexadecane interface is reported. Similar to the water/air interfaces, the microcubes were shown to reside at the water/hexadecane interface with three distinct orientations: face-up, edge-up, and vertex-up. Concomitantly, ordered aggregates with flat plate, tilted linear, and close-packed hexagonal structures were formed, driven by capillary force. With increasing the hydrophobicity of five sides of the cubes, the preferential microcube orientation at the water/hexadecane interface changed sequentially from face-up to edge-up, to vertex-up, then back to edge-up, and to face-up. This dependence of the preferential microcube orientation on surface hydrophobicity at the water/hexadecane interface differs from that observed at the water/air interface, where the preferential orientation changed only from face-up to edge-up, then to vertex-up, as surface hydrophobicity increased. In addition, preformed microcube assemblies at the water/air interface could be dynamically reconfigured by replacing the air phase with hexadecane under stirring.