<p>Diffraction of matter waves is a fundamental consequence of quantum mechanics, directly illustrating the core principles of wave-particle duality, quantum superposition, and quantum interference. De Broglie’s proposal that particles exhibit wave-like properties has been experimentally confirmed for electrons, neutrons, and composite systems such as helium atoms, molecules and clusters. Here, we report the observation of positronium diffraction using a high-quality, energy-tunable positronium beam transmitted through graphene. Time-of-flight selection and spatially resolved detection reveal a distinct 1st-order diffraction peak at a position consistent with the prediction from matter-wave considerations for positronium. This work provides the direct and definitive evidence of quantum interference in positronium beams and confirms that it behaves as a single quantum entity rather than two independent interfering particles. This groundbreaking experimental milestone marks a major advance in fundamental physics, not only demonstrating positronium’s wave nature as a bound lepton-antilepton system but also opening pathways for precision measurements involving positronium.</p>