The diamondback moth, Plutella xylostella, the main pest of brassica crops, has developed resistance to almost all major classes of insecticides as the farmers rely on insecticides to control this pest. An extensive use of broad-spectrum insecticides against P. xylostella promotes the selection of insecticide resistance, destroy natural enemies, and pollute the environment. In this scenario, it is imperative to use genetic methods such as gene silencing technology as an alternate approach against this pest. Evidence shows that microRNAs play pivotal roles in the regulation of target genes at the post-transcription level and show differential expression under various biological processes. However, the knowledge of their role in insect immunity is still in its infancy. In the present study, we aimed at exploring the response of P. xylostella miRNAs against B. thuringiensis at different time courses (6, 12, 18, 24, and 36 h) by using small RNA sequencing. After data filtration, a combined set of 149 miRNAs was identified from all the libraries. Interestingly, a couple of conserved miRNAs such as miR-1, Let-7, miR-275, miR-184, and miR-10 were listed as abundantly expressed miRNAs after exposure to B. thuringiensis. It is worth mentioning that the differential expression analysis revealed that miR-2, a conserved miRNA, was up-regulated following infection. Furthermore, we experimentally validated the involvement of miR-2b-3p in the regulation of corresponding target trypsin. Our luciferase assay results revealed that miR-2b-3p mimic significantly down-regulated the target gene trypsin indicating that it might play a crucial role in the defense mechanism of P. xylostella against B. thuringiensis infection. On the whole, our findings provide insights into the possible regulatory role of miRNAs in insect immunity in response to microorganisms.