Electron flying qubits are envisioned as potential information links within a quantum computer, but also promise-like photonic approaches-to serve as self-standing quantum processing units. In contrast to their photonic counterparts, electron-quantum-optics implementations are subject to Coulomb interactions, which provide a direct route to entangle the orbital or spin degree of freedom. However, controlled interaction of flying electrons at the single-particle level has not yet been established experimentally. Here we report antibunching of a pair of single electrons that is synchronously shuttled through a circuit of coupled quantum rails by means of a surface acoustic wave. The in-flight partitioning process exhibits a reciprocal gating effect which allows us to ascribe the observed repulsion predominantly to Coulomb interaction. Our single-shot experiment marks an important milestone on the route to realize a controlled-phase gate for in-flight quantum manipulations.