Synaptobrevin (Syb)/vesicle-associated membrane protein (VAMP) is a small, integral membrane protein of synaptic vesicles. Two homologous isoforms of synaptobrevin, Syb1/VAMP1 and Syb2/VAMP2, exhibit distinct but partially overlapping patterns of expression in adult mammalian neurons: Syb1 is predominantly expressed in the spinal cord, especially in motor neurons and motor nerve terminals of the neuromuscular junction (NMJ), whereas Syb2 is primarily expressed in central synapses in the brain. Whereas many studies have focused on the function of Syb2 in the brain, few studies have examined the role of Syb1. Here we report that Syb1 plays a critical role in neuromuscular synaptic transmission. A null mutation of Syb1 resulting from a spontaneous, nonsense mutation in mice significantly impairs the function, but not the structure, of the NMJ. In particular, both spontaneous and evoked synaptic activities in Syb1 mutant mice are reduced significantly relative to control mice. Short-term synaptic plasticity in Syb1-deficient NMJs is markedly altered: paired-pulse facilitation is significantly enhanced, suggesting a reduction in the initial release probability of synaptic vesicles. Furthermore, Syb1-deficient NMJs display a pronounced asynchrony in neurotransmitter release. These impairments are not due to an alteration of the size of the readily releasable pool of vesicles, but are attributable to reduced sensitivity and cooperativity to calcium (Ca2+) due to the absence of Syb1. Our findings demonstrate that Syb1 plays an essential, non-redundant role in Ca2+-triggered vesicle exocytosis at the mouse NMJ.