Phonon lasers are mechanical analogues of the ubiquitous optical laser and have been realized in a variety of contexts1–12. However, no such demonstration exists for mesoscopic levitated optomechanical systems, which are emerging as important platforms for conducting fundamental tests of quantum mechanics13–15 and gravity16, as well as for developing sensing modalities that couple mechanical motion to electron spin17–20 and charge21. Inspired by the pioneering work of Arthur Ashkin on optical tweezers22,23, we introduce a mesoscopic, frequency-tunable phonon laser based on the centre-of-mass oscillation of a silica nanosphere levitated in an optical tweezer under vacuum. Unlike previous levitated realizations, our scheme is general enough to be used on single electrons, liquid droplets or even small biological organisms24. Our device thus provides a pathway for a coherent source of phonons on the mesoscale that can be applied to both fundamental problems in quantum mechanics as well as tasks of precision metrology25–27. A phonon laser based on an optically levitated silica nanosphere is demonstrated. A lasing threshold—a phase transition from Brownian motion to coherent oscillation—is observed when the modulation depth of the trapping beam power is increased.