Folate metabolism pathway is mainly associated with syntheses of nucleic acids and proteins as well as DNA methylation and repair. Cytoplasmic C1 tetrahydrofolate (C1-THF) synthase is a central enzyme in folate metabolism and plays an important role in C1-THF conversion. The full-length sequence of silkworm (Bombyx mori) cytoplasmic C1-THF synthase (BmC1-THF synthase) gene including an open reading frame (ORF) of 932 amino acid residues was cloned. Phylogenetic analysis indicated that BmC1-THF synthase shares 84% and 81% identity with C1-THF synthase of Papilio Xuthus and Danaus plexippus, respectively. Conserved sequence analysis showed that the 330 to 710 amino acid residues of BmC1-THF synthase were highly conserved among various organisms. Recombinant BmC1-THF synthase protein with a molecular weight of about 105.5 kDa was expressed in E. coli cells and identified using SDS-PAGE and western blot analysis. The expression of BmC1-THF synthase gene in eight silkworm tissues revealed that BmC1-THF synthase gene is widely expressed in various tissues of silkworm and highly expressed in the fat body. The expression characteristics of BmC1-THF synthase gene in response to oxidative stress were conducted under phoxim and high temperature (30 °C) exposure. The results showed that the transcriptional level of BmC1-THF synthase gene was upregulated by 3.435-fold and 6.845-fold after phoxim and high temperature (30 °C) exposure, respectively, indicating that BmC1-THF synthase gene may play a significant role in response of silkworm to oxidative stresses.