Glutaminase-free l-asparaginase purified from Trichoderma viride culture filtrate, with 36 purification folds, 688.1 U/mg specific activity, and 38.9% yield, has been found to exert statistically significant high antiproliferative activity against hepatocellular carcinoma both in vitro using Hep-G2 cell line, with an IC50 of 21.2 g/mL, and in vivo using DENA intoxicated murine model, where the latter showed a significant resolution of the hepatocellular damage approaching the healthy control group. This evidence concerns the gene ASPG and hepatocellular carcinoma.