Transcriptomic profiles of the serious lepidopteran insect pest Lymantria dispar (gypsy moth) were characterized in the larval midgut in response to infection by Bacillus thuringiensis kurstaki, a biopesticide commonly used for its control. RNA-Seq approaches were used to define a set of 49,613 assembled transcript sequences, of which 838, 1,248 and 3,305 were respectively partitioned into high-, mid- and low-quality tiers on the basis of homology information. Digital gene expression profiles suggested genes differentially expressed at 24 hours post infection, and qRT-PCR analyses were performed for verification. The differentially expressed genes primarily associated with digestive function, including α-amylase, lipase and carboxypeptidase; immune response, including C-type lectin 4; developmental genes such as arylphorin; as well as a variety of binding proteins: cellular retinoic acid binding protein (lipid-binding), insulin-related peptide binding protein (protein-binding) and ovary C/EBPg transcription factor (nucleic acid-binding). This is the first study conducted to specifically investigate gypsy moth response to a bacterial infection challenge using large-scale sequencing technologies, and the results highlight important genes that could be involved in biopesticide resistance development or could serve as targets for biologically-based control mechanisms of this insect pest.