INTRODUCTION: Oxygen (O2 ) nanobubbles offer a new method for tissue oxygenation. The effects of O2 nanobubbles on transmission at neuromuscular junctions (NMJs) and muscle function were explored in murine diaphragm. METHODS: Electrophysiological parameters, NMJ ultrastructure, muscle force, and muscle fatigue were studied during superfusion with solutions with different oxygen levels or oxygen nanobubbles. RESULTS: High frequency nerve stimulation of muscles superfused with O2 nanobubble solution slowed neurotransmission decline over those with either control or hyperoxic solution. O2 nanobubble solution increased the amplitude of evoked end plate potentials and quantal content but did not affect spontaneous activity. Electron microscopy of stimulated O2 nanobubble treated NMJs showed accumulation of large synaptic vesicles and endosome-like structures. O2 nanobubble solution had no effects on isometric muscle force, but it significantly decreased fatigability and maximum force recovery time in nerve stimulated muscles. CONCLUSIONS: O2 nanobubbles increase neurotransmission and reduce the probability of neurotransmission failure in muscle fatigue. Muscle Nerve 55: 555-563, 2017.