Tumstatin45-132 is an 88-amino-acid fragment possessing the equivalent ability of full-length tumstatin to block new tumour blood-vessel formation and suppress tumour growth. TNFalpha (tumour necrosis factor alpha), an antitumour agent, is used in clinical therapy, but is limited by its strong systemic toxicity. Combining TNFalpha with tumstatin45-132 may represent a promising alternative approach in cancer therapy. In the present study, we expressed recombinant tumstatin45-132-TNFalpha in a baculovirus expression system. We evaluated the effects of tumstatin45-132-TNFalpha on neovascularization and cell viability by a chick-embryo-chorioallantoic-membrane assay and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. In-vivo antitumour activities were examined in tumour-bearing mice. We observed that tumstatin45-132-TNFalpha inhibited angiogenesis and tumour-cell viability in vitro. In-vivo experiments showed that intratumoural injection of tumstatin45-132-TNFalpha significantly inhibited the growth of xenograft tumours in mice. MRI analysis revealed that tumstatin45-132-TNFalpha treatment also decreased mean blood-vessel density in vivo. Tumstatin45-132-TNFalpha exerted antitumour activities by decreasing proliferation, inducing apoptosis in tumour cells and anti-angiogenesis. In conclusion, our findings suggest that tumstatin45-132-TNFalpha has significant activity against F6 tumour cells and that it may be a potential approach for cancer therapy.