Lysine succinylation is a newly identified protein post-translational modification (PTM) of lysine residues. Increasing evidences demonstrate that this modification is prevalent in mitochondria and regulates many vital cellular processes, especially metabolism. Here, we determined the succinylome of the silkworm (Bombyx mori) midgut mitochondria during the larval gluttonous stage (the fifth instar) using succinylated peptides enrichment coupled with nano HPLC/MS/MS. A total of 1884 lysine succinylation sites on 373 mitochondrial proteins were identified. The bioinformatic analysis reveal that succinylated proteins are significantly enriched in central metabolic processes and mitochondrial protein synthesis. Several apoptosis and detoxification related enzymes or proteins are succinylated. The findings suggest the crucial role of lysine succinylation in silkworm midgut metabolism and resistance. Our data provide a rich resource for further analysis of lysine succinylation in silkworm.Insect midgut is the vital tissue for nutrient metabolism and also for xenobiotic metabolism. There is a growing body of knowledge on regulation of midgut function at the gene or protein levels in silkworm, however, the regulation at post-translation modification level remains largely unknown. We provide a first global analysis of the mitochondrial lysine succinylome in silkworm midgut. A total of 1884 lysine succinylation sites on 373 mitochondrial proteins were identified. Bioinformatics results suggest an important role of this modification in regulating metabolism and mitochondrial protein synthesis. Our data greatly expand the catalog of lysine succinylation substrates and sites in insects, and represents an important resource for understanding the physiological function of lysine succinylation in insect midgut.