Matter-wave interferometers provide an opportunity to measure whether quantum superpositions exist at macroscopic length scales or only at microscopically small scales; now such instruments have demonstrated quantum interference of wave packets separated by 54 cm. Matter-wave interferometers, which allow for the observation of interference pattern of atomic wave packets that are split and recombined, have proven to be useful tools in precision metrology and fundamental research. These interferometers provide an opportunity to measure whether quantum superpositions exist at macroscopic length scales or only at microscopically small scales. But a truly macroscopic scale that would be necessary for such a test had not been reached in matter-wave interferometers to date. Here the authors show quantum interference of wave packets separated by 54 cm. Their matter-wave interferometer also promises increased sensitivity in precision tests, for example, when measuring the equivalence principle or measuring gravity. The quantum superposition principle allows massive particles to be delocalized over distant positions. Though quantum mechanics has proved adept at describing the microscopic world, quantum superposition runs counter to intuitive conceptions of reality and locality when extended to the macroscopic scale1, as exemplified by the thought experiment of Schrodinger’s cat2. Matter-wave interferometers3, which split and recombine wave packets in order to observe interference, provide a way to probe the superposition principle on macroscopic scales4 and explore the transition to classical physics5. In such experiments, large wave-packet separation is impeded by the need for long interaction times and large momentum beam splitters, which cause susceptibility to dephasing and decoherence1. Here we use light-pulse atom interferometry6,7 to realize quantum interference with wave packets separated by up to 54 centimetres on a timescale of 1 second. These results push quantum superposition into a new macroscopic regime, demonstrating that quantum superposition remains possible at the distances and timescales of everyday life. The sub-nanokelvin temperatures of the atoms and a compensation of transverse optical forces enable a large separation while maintaining an interference contrast of 28 per cent. In addition to testing the superposition principle in a new regime, large quantum superposition states are vital to exploring gravity with atom interferometers in greater detail. We anticipate that these states could be used to increase sensitivity in tests of the equivalence principle8,9,10,11,12, measure the gravitational Aharonov–Bohm effect13, and eventually detect gravitational waves14 and phase shifts associated with general relativity12.