Tick saliva contains a broad array of secretory products facilitating prolonged tick attachment and feeding, and is the usual conduit for pathogens into hosts. Protein secretion into saliva from tick salivary glands is due to exocytosis of vesicular membrane-bound granular material regulated by SNARE [soluble N-ethylmaleimide-sensitive factor attachment protein receptor] complex proteins. Proteins associated with vesicles, cytoplasm and target membranes are essential components of the exocytotic machinery in tick salivary glands, and many of the genes encoding those proteins have been identified. One of these genes is homologous to the N-ethylmaleimide sensitive fusion factor (NSF), whose protein plays an important role in vesicle fusion in other eukaryotic cells. We investigated the NSF homolog gene present in the salivary gland of two tick species, Ixodes scapularis and Dermacentor variabilis, and evaluated the functional role of NSF in both ticks using in vivo RNA interference. The salivary NSF gene was disrupted by injecting adult ticks with 500 ng of dsRNA complementing the gene sequence. Silencing was demonstrated by reduced transcript levels using qPCR and RT-PCR in midguts and salivary glands. Disrupting expression of NSF by RNAi completely reduced the ability of ticks to feed on NZ white rabbits. These studies demonstrate that NSF plays a vital role in salivary protein secretion. The potential role of NSF in intracellular pathogen trafficking through tick salivary glands also will be discussed.