Toll-like receptors (TLRs) are the critical superfamily homologues that initiate sensing of the invasion of pathogens by the Toll pathway. As one of several intracellular nucleic acid-sensing TLRs, TLR13 is activated by an unmethylated motif present in the large ribosomal subunit of bacterial RNA. However, little attention has been paid to the function of TLR13 gene homologue from Laodelphax striatellus (designated as LsToll-13) in the immune response to rice stripe virus (RSV). Herein, LsToll-13 was cloned and characterized using RACE-PCR. Phylogenetic analysis showed that LsToll-13 was clustered with the TLR13 from six insects. Real-time PCR analysis demonstrated that the expression level of LsToll-13 was significantly reduced in L. striatellus with RSV infection compared with that in the naive strain. When the expression of LsToll-13 was significantly up-regulated at 6 h after bacterial infection, the expression of ribonucleoprotein (RNP) indicated that the RSV titer in the host insect was significantly suppressed. Upon knockdown of LsToll-13, using RNA interference (RNAi) in L. striatellus, the expression level of RNP was significantly increased with enhanced RSV accumulation, suggesting that LsToll-13 potentially protects L. striatellus from RSV infection. Taken together, our results indicated that LsToll-13 might be involved in the immune response of L. striatellus to RSV infection, and provided a new insight into further elucidating the molecular mechanisms of complex pathogen-host interactions and integrative pest management.