<p>Zinc (Zn²⁺) is the second most abundant trace element after iron, with most of it is&#xa0;stored in skeletal muscles. Although a large part of Zn²⁺ is tightly bound to metalloproteins, the small portion of free Zn²⁺ can participate in nerve signaling. Here we examined the effects of Zn²⁺ at nanomolar concentrations on neuromuscular transmission in the diaphragm, the main respiratory muscle. Zn²⁺ reduced spontaneous neurotransmitter release at both lowered and physiological external Ca²⁺ levels. Additionally, Zn²⁺ effectively decreased the probability of neurotransmitter release upon single nerve stimulation under lowered external Ca²⁺, and inhibited Ca²⁺-independent sucrose-induced exocytosis. At physiological external Ca²⁺ concentration, Zn²⁺ decreased neurotransmitter release during low-frequency stimulation. The reduction became increased during short trains of moderate-to-high frequency stimuli. Furthermore, Zn²⁺ diminished both neurotransmitter release and the participation of dye-labeled synaptic vesicles in exocytosis during prolonged nerve firing at moderate frequency. Zn²⁺ aggravated muscle fatigue and impaired contraction recovery upon nerve stimulation. This was linked to a reduction in peak inspiratory flow in mice, an indicator of diaphragm function, after injection of low-dose Zn²⁺. Our data suggest that at nanomolar concentrations, Zn²⁺ is a negative modulator of neuromuscular function.</p>