The Japanese pine sawyer, Monochamus alternatus Hope (Coleoptera: Cerambycidae), is a major pest of coniferous forests. Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) (Bt) is an ecosystem-friendly agent for the control of pest insects. To identify the genes that are potentially associated with Bt toxicology, differentially expressed gene (DGE) libraries of M. alternatus after 20 h of exposure to a sublethal concentration of Bt were prepared from dissected midgut tissue. The transcripts were sequenced using the Illumina sequencing platform. After quality checks, the total numbers of clean reads were 29.20 and 41.53 million in the control and Bt-exposed libraries, respectively. We found that 947 and 8,895 unique genes were significantly up- and down-regulated, respectively. The annotation of the non-redundant sequences using the Gene Ontology database identified 3,837 sequences involved in cellular components, 3,190 sequences involved in molecular functions, and 6,930 sequences involved in biological processes, and showed that the genes were distributed across 50 categories. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that the top 20 enriched pathways included metabolism of xenobiotics by the cytochrome P450 pathway and steroid hormone biosynthesis. Seventy-four DEGs were enriched in the metabolism of xenobiotics by the cytochrome P450 pathway, including the DEGs encoding Cyp1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), Cyp1A2 (cytochrome P450, family 1, subfamily A, polypeptide 2), Cyp3A (cytochrome P450, family 3, subfamily A), aldehyde dehydrogenase (NAD[P] ) and ADH1_7 (alcohol dehydrogenase 1/7), all of which were up-regulated. However, the DEGs encoding cathepsin L and CBR1 (carbonyl reductase 1) were down-regulated. Therefore, the effects of Bt on M. alternatus are complex and time dependent. This study is a first step toward achieving an understanding of the profile of the secondary targets of Bt in M. alternatus, and the results may provide insights for further exploration of the functions of the target genes in detoxification and resistance to Bt.