After a long pause, the accumulation of data on the involvement of tumor-specific DNA and extracellular DNA in metastasis has again placed enzymes with deoxyribonuclease activity in the focus of the search for antitumor and antimetastatic drugs. In this work, the ability of bovine pancreatic DNase I to reduce the invasive potential of B16 melanoma has been investigated in vitro and in vivo. It was found that DNase I had a cytotoxic effect on B16 melanoma cells (IC₅₀ ≈ 10⁴ U/mL). At the same time, significantly lower doses of DNase I (10²-10³ U/mL) inhibited the migratory activity of melanoma cells in vitro, causing a decrease in the distance of cell front migration and in the area of scratch healing 48 h after the enzyme addition, as well as reducing the rate of cell migration. In mice with B16 metastatic melanoma, intramuscular administration of DNase I in the dose range of 0.12-1.20 mg/kg resulted in a two- to threefold decrease in the number of surface lung metastases and caused nonspecific antigenic immune stimulation.