Hepatocellular carcinoma (HCC) is a representative inflammation-associated cancer and known to be the most frequent tumor. However, the preventive agents for hepatocarcinogenesis are unsatisfactorily identified. We investigated the protective effect of steamed and freeze-dried mature silkworm larval powder (SMSP) on diethylnitrosamine (DEN)-induced hepatotoxicity in mice and compared the effect of three silkworm varieties: white-jade, golden-silk, and light-green strains. The mice were fed with diet containing 0.1, 1, and 10 g/kg of three types of SMSP for two weeks while DEN (100 mg/kg, i.p.) was injected 18 h before the end of this experiment. Liver toxicity was determined as serum indicator, histopathological examination, and expression of inflammatory enzyme. Pretreatment with SMSP reduced necrotic and histopathological changes induced by DEN in the liver. The measurement of serum biochemical indicators showed that pretreatment with SMSP also decreased DEN-induced hepatotoxicity, the levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). In addition, SMSP inhibited the expressions of inflammatory enzymes, cyclooxygenase-2 and inducible nitric oxide synthase. White-jade SMSP showed the most effective hepatoprotective results against hepatotoxicity among the three silkworm strains used in this study. SMSP may have a protective effect against acute liver injury by inhibiting necrosis and inflammatory response in DEN-treated mice.