Trichinella spiralis(T.spiralis) adult-specific deoxyribonuclease II-7 (TsDNase II-7), a member of the DNase II-like nuclease family with no DNase II activity, was identified in the excretory–secretory (ES) products of adult worms (AWs). However, its biological functions are still unclear. Our previous study revealed thatTsDNase II-7 is located around the infection site in the intestinal tissue, speculating that it was involved in theT.spiralisinvasion of host intestinal epithelial cells (IECs). This study aimed to use RNA interference to verify our speculation thatTsDNase II-7 in 3-day old adultT.spiralis(Ad3) plays a role in intestinal invasion.TsDNase II-7-specific small interfering RNAs (siRNAs) were delivered into muscle larvae (MLs) to knockdownTsDNase II-7 expression by electroporation. Twenty-four hours later, the MLs transfected with 2 μM siRNA-841 exhibited decreased inTsDNase II-7 transcription and expression as compared to the control MLs. The knockdown ofTsDNase II-7 expression did not affect ML viability, and the low expression ofTsDNase II-7 still maintained in Ad3 recovered fromTsDNase II-7-RNAi-ML infected mice, resulting in a weakened ability of Ad3 to invade intestinal epithelial cells (IECs). These results indicated that knockdown ofTsDNase II-7 gene expression via RNA interference (RNAi) suppressed adult worm invasion and confirmed thatTsDNase II-7 plays a crucial role during the intestinal phase ofT.spiralisinfections, which provided new candidate for vaccine development ofT.spiralis.