Oncolytic viruses are a promising tool for treatment of cancer. We studied an oncolytic Semliki Forest virus (SFV) vector, VA7, carrying the enhanced green fluorescent protein gene (EGFP), as a novel virotherapy candidate against unresectable osteosarcoma. The efficiency and characteristics of the VA7-EGFP treatment were compared with a widely studied oncolytic adenovirus, Ad5Delta24, both in vitro and in vivo. VA7-EGFP resulted in more rapid oncolysis and was more efficient at low multiplicities of infection (MOI) when compared with Ad5Delta24 in vitro. Yet, in MG-63 cells, a subpopulation resistant to the VA7-EGFP vector emerged. In subcutaneous human osteosarcoma xenografts in nude mice treatment with either vector reduced tumor size, whereas tumors in control mice expanded quickly. The VA7-EGFP-treated tumors were either completely abolished or regressed to pinpoint size. The efficacy of VA7-EGFP vector was studied also in an orthotopic osteosarcoma nude mouse model characterized by highly aggressive tumor growth. Treatment with oncolytic SFV extended survival of the animals significantly (P < 0.01), yet none of the animals were finally cured. Sera from SFV-treated mice contained neutralizing antibodies, and as nude mice are not able to establish IgG response, the result points out the role of IgM class antibodies in clearance of virus from peripheral tumors. Furthermore, biodistribution analysis at the survival end point verified the presence of virus in some of the brain samples, which is in line with previous studies demonstrating that IgG is required for clearance of SFV from central nervous system.