ABSTRACT: Insect C-type lectins (CTLs) play vital roles in modulating humoral and cellular immune responses. The oriental armyworm, Mythimna separata (Walker) (Lepidoptera: Noctuidae) is a migratory pest that causes significant economic loss in agriculture. CTLs have not yet been systematically identified in M. separata. In this study, we first constructed a transcriptome of M. separata larvae, generating a total of 45,888 unigenes with an average length of 910 bp. Unigenes were functionally annotated in six databases: NR, GO, KEGG, Pfam, eggNOG, and Swiss-Prot. Unigenes were enriched in functional pathways, such as those of signal transduction, endocrine system, cellular community, and immune system. Thirty-five unigenes encoding C-type lectins were identified, including CTL-S1~CTL-S6 (single CRD) and IML-1~IML-29 (dual CRD). Phylogenetic analyses showed dramatic lineage-specific expansions of IMLs. Sequence alignment and structural modeling identified potential ligand-interacting residues. Real-time qPCR revealed that CTL-Ss mainly express in eggs and early stage larvae, while IMLs mainly express in mid-late-stage larvae, pupae, and adults. In naïve larvae, hemocytes, fat body, and epidermis are the major tissues that express CTLs. In larvae challenged by Escherichia coli, Staphylococcus aureus, or Beauveria bassiana, the expression of different CTLs was stimulated in hemocytes, fat body and midgut. The present study will help further explore functions of M. separata CTLs.