As a pathogenic bacterium, Bacillus thuringiensis (Bt) has become an alternative to chemical insecticides in commercial agricultural to control forestry pests and mosquitoes. To prevent pest resistance, many novel Bt strains have been isolated. Strain S3580-1 (WGS: VHPX0000000) used in this research and originally isolated from Hainan Qixianling National Forest Park (China) showed significant toxicity to Culex pipiens pallens. Here, using whole genome sequencing, assembly, and bioinformatics analysis, the predicted S3580-1CG_5163 (GenBank Accession No. MK124137) gene-encoded protein was found to share low homology with known toxins designated by the Bt toxin nomenclature system. It was considered to be an ETX/MTX2-type toxin and was designated Epp. Bioinformatics analysis showed that the predicted S3580-1CG_5163 gene-encoded protein Epp shared low identity with other known toxic protein sequences containing Cry-ETX/MTX conserved domains at the amino acid level, but significant similarity at the structural level. In addition, bioassays showed that Epp was toxic against Spodoptera litura (LC 296.133 μg/mL; 95% FL 200.555-471.318 μg/mL) and Cx. pipiens pallens (LC 322.193 μg/mL; 95% FL 238.217-477.243 μg/mL). On pathological observation, the peritrophic membrane of Cx. pipiens pallens larvae was degraded causing the midgut structure to become incomplete, resulting in larval death. Further bioassays are required to fully elucidate the insecticidal spectrum of the ETX/MTX2-type toxin Epp, and thereby provide future research directions.