Globally, the edible insect industry is emerging due to its potential contributions to food security and environmental sustainability. Edible insects are rapidly being integrated into the development of alternative foods and new pharmaceuticals. Silkworms, known for their high protein content, are not only a potential new source of human food and animal feed but have also been traditionally used for medicinal purposes. However, conventional silkworms are difficult to ingest. To address this, we have developed a steamed and freeze-dried mature silkworm larva powder (SMSP), and it is essential to investigate its potential toxicity and food safety for further studies and applications. Therefore, this study aimed to evaluate the toxicity of SMSP. A toxicity assessment of SMSP was conducted according to OECD guidelines. An oral repeat-administration study was performed on male and female SD rats at doses of 625, 1250, and 2500 mg/kg/day for 4 and 13 weeks. No toxicological changes were observed in clinical signs, body weight, water and food intake, urine tests, hematology, clinical biochemistry, gross findings, or histopathological examination. In conclusion, the no observed adverse effect level (NOAEL) of SMSP was 2500 mg/kg/day, with no target organs identified in either sex of the rats. These results suggest that SMSP is safe, is without side effects and has potential for use as an edible ingredient and in health functional food applications.