ABSTRACTRNA interference (RNAi) is a specific post-transcriptional gene-silencing phenomenon, which plays an important role in the regulation of gene expression and the protection from transposable elements in eukaryotic organisms. InDrosophila melanogaster, RNAi can be induced by microRNA (miRNA), endogenous small interfering RNA (siRNA), or exogenous siRNA. However, the biogenesis of miRNA and siRNA in these RNAi pathways is aided by the double-stranded RNA binding proteins (dsRBPs) Loquacious (Loqs)-PB, Loqs-PD or R2D2. In this study, we identified three alternative splicing variants ofLoqs, namelyLoqs-PA, -PB, and -PCin the orthopteranLocusta migratoria. We performedin vitroandin vivoexperiments to study the roles of the three Loqs variants in the miRNA- and siRNA-mediated RNAi pathways. Our results show that Loqs-PB assists the binding of pre-miRNA to Dicer−1 to lead to the cleavage of pre-miRNA to yield matured miRNA in the miRNA-mediated RNAi pathway. In contrast, different Loqs proteins participate in different siRNA-mediated RNAi pathways. In exogenous siRNA-mediated RNAi pathway, binding of Loqs-PA or LmLoqs-PB to exogenous dsRNA facilitates the cleavage of dsRNA by Dicer−2, whereas in endogenous siRNA-mediated RNAi pathway, binding of Loqs-PB or Loqs-PC to endogenous dsRNA facilitates the cleavage of dsRNA by Dicer−2. Our findings provide new insights into the functional importance of different Loqs proteins derived from alternative splicing variants ofLoqsin achieving high RNAi efficiency in different RNAi pathways in insects.