Background: A major obstacle to the oncolytic virus treatment approach has been the rapid virus elimination by patient’s immune system. We hypothesized that oncolytic viruses would be protected and delivered efficiently to tumor sites by autologous adipose stromal vascular fraction (SVF) cells. Here, we report the results of a study to determine the safety and feasibility of this approach in cancer patients. Methods: In this single-arm, open-label safety study, 24 patients with advanced solid tumors and 2 patients with AML were treated with a single administration of the oncolytic virus ACAM2000 (vaccinia) delivered by SVF cells. Patients received ACAM2000/SVF by intravenous application, or by a combination of intravenous and intratumoral or intra-peritoneal injections. The primary endpoint was safety/tolerability. Secondary endpoints assessed overall survival and induction of immune responses. Blood samples were collected at multiple time points for quantifying vaccinia virus DNA in peripheral blood and to determine the plasma cytokine levels and the effects on several immune cell types. Results: No serious toxicities were reported. Eight subjects reported an AE: self-limiting skin lesions, lasting 7 to 18 days. No infusion-related AEs were reported. No AEs leading to study discontinuation were reported. Viral DNA was detected in all patients immediately following treatment. Interestingly, in 8 patients viral DNA disappeared 1 day and re-appeared 1 week post treatment, suggesting active viral replication, possibly at tumor sites. This viral DNA reappearance correlated with longer survival of these patients. No major increase in cytokine levels was observed in any of the patients. No correlation between cytokine levels and skin lesions was noted. Flow cytometry showed gradual changes suggesting improved immune cell activation status. Tumor size reduction was documented in several patients. Conclusions: Treatment with ACAM2000/SVF in patients with advanced solid tumors and AML is safe and well tolerated, with clear antitumor effects in several patients. These promising initial clinical results merit further investigation of therapeutic utility. Clinical trial information: ISRCTN#10201650.