The intense dust devils and dust storms on Mars are believed to generate large electrostatic fields that significantly alter geophysical and geochemical processes on the planet. The existence of such fields must be related to a mechanism by which charged dust separates by polarity; it has been widely hypothesized that this separation originates from a particle-size dependence of the charge polarity, but this effect has never been demonstrated. To address this issue, we carry out experiments on the triboelectric charging of Martian regolith simulant (JSC-1 Mars), using a fluid flow apparatus wherein only particle-particle interactions occur, as is the case in Martian dust events. Our experiments show direct evidence that smaller particles tend to charge negatively and larger particles tend to charge positively, which provides a mechanism for the charge separation that creates electric fields in Martian dust events.