Background: We investigated possible role of the NF-kB in the anticancer effect of nitroglycerin with metformin on experimental BHK-21/C13-induced fibrosarcoma in hamsters. Material and Methods: Peroral treatment of tumor-bearing hamsters carried out with a pure combination of NF-kB suppressors nitroglycerin 25 mg/kg and metformin 500 mg/kg daily and with additional rescue doses of NF-kB stimulator mebendazole 460 mg/kg daily, via a gastric probe after tumor inoculation. After animal sacrification, blood samples were collected for hematological and biochemical analyses, the tumors were excised and weighed, and their diameters and volumes were measured. The tumor samples were pathohistologically and immunohistochemically assessed for proliferation marker protein Ki-67, proliferating cell nuclear antigen PCNA, hematopoietic progenitor cell antigen CD34, cluster of differentiation 31 (CD31), cytochrome c oxidase subunit 4 (COX4), mitochondria marker Cytochrome C, glucose transporter 1 (GLUT1) and inducible nitric oxide synthase (iNOS), and the main organs were toxicologically tested. The Ki-67 and PCNA positivity and the cytoplasmic marker (CD34, CD31, COX4, Cytochrome C, GLUT1, iNOS) immunoexpression in the tumor samples were quantified. Results: The combination of NF-kB suppressors nitroglycerin and metformin significantly inhibited fibrosarcoma growth in hamsters without toxicity, compared to control. Co-treatment with NF-kB stimulator mebendazole inhibited anticancer activity of the NF-kB suppressors nitroglycerin and metformin combination. NF-kB stimulator mebendazole rescued tumor progression inhibited by the NF-kB suppressors nitroglycerin and metformin. Conclusion: Anticancer effect of nitroglycerin with metformin might be through NF-kB suppression and might be an effective and safe approach in novel nontoxic adjuvant and relapse prevention oncological treatment.