Our previous works confirmed that RNAi efficiency can be achieved by injection of dsRNA into the fourth-instar larva or pupa in Spodoptera litura. However, it has been considered that the RNAi efficiency is refractory in lepidopteran insects. In the present study, we focused on the RNAi machinery in S. litura by using an our previously constructed transcriptome (GenBank TSA: GBBY00000000) to detect genes predicted to be involved in the RNAi response, including the R2d2, Loqs, Ago1, Ago2, Dic1 and Dic2. By using quantitative PCR, we found that these genes were highly expressed in the development stage of adult and in the tissue of fat body. We also found three homologues of the sid1-like gene responsible for dsRNA transport inside the cell in the S. litura transcriptome. These genes were preferentially expressed in the pupal stage and in the tissue of midgut. Z-test of neutral evolution analysis showed that only between Sl-sid1 and Sl-sid3 have the probability of rejecting the null hypothesis of strict-neutrality (dN = dS, P< 0.05), indicating no evolutionary pressure between Sl-sid1 and Sl-sid3. Our data support that S. litura may have a conserved RNAi mechanism, but that different tissues and/or developmental stages may differ in the efficacy of the RNAi response.