In addition to serving as the means for introducing pathogens into hosts, tick saliva contains a broad array of secretory products that facilitate prolonged tick attachment and feeding. Because ticks transmit a broad array of disease-causing agents, disrupting tick blood feeding or inactivating tick salivary proteins presents a novel strategy for tick-borne disease prevention. Cystatin, a salivary cysteine proteinase inhibitor, may be a key target for such an intervention. Cystatin is expressed in Amblyomma americanum salivary glands and secreted in tick saliva during the blood feeding process. RNA interference (RNAi) was used to assess the role of this molecule for tick feeding success. The salivary cystatin gene was silenced by injecting adult ticks with 500 ng of dsRNA complementing the gene sequence. Silencing was demonstrated by reduced transcript and protein levels in salivary glands removed from partially-fed ticks. Disrupting expression of cystatin by RNAi reduced the ability of ticks to feed successfully, as demonstrated by prolonged attachment times and reduced engorgement weights. These studies demonstrate that RNAi can be a useful tool for identifying functionally important tick salivary gland products.