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Entomopathogenic nematodes (EPN) are biological control agents used to control different insect pests. Indigenous EPN resources are important for the successful control of endemic pests. A native EPN isolate collected in Yunnan, China that showed high virulence against Spodoptera litura was identified and studied for virulence and production potential. The EPN isolate was identified as Steinernema ceratophorum and showed high virulence against different larval stages of Spodoptera exigua, Ostrinia nubilalis, Helicoverpa armigera, and the 2nd instar larvae of Spodoptera frugiperda when applied at 50 IJ/larva under in vitro conditions. In vivo culture of the S. ceratophorum isolate showed that Galleria mellonella larvae were the most suitable host compared to Tenebrio molitor and S. litura. The highest yield in in vivo culture was 1.15 × 105 IJ/larva when cultured in G. mellonella with inoculum concentration of 50 IJ/larva at 25°C. For in vitro solid culture, the highest yield for S. ceratophorum was 2.09 × 106 IJ/g when cultured at optimal temperature of 25°C. Nematodes from the in vivo and in vitro culture, both retained their virulence against the G. mellonella larvae. The S. ceratophorum showed high potential to be mass produced, which will enable the commercial use of this EPN isolate. This is the first attempt to mass culture the S. ceratophorum species. The study explores the usage of S. ceratophorum and provides basis for the commercialization of the EPN species.