Peritoneal dissemination is a common and fatal clinical manifestation of gastric cancer with few effective therapies available. Natural Newcastle disease virus (NDV) has been shown to be an effective oncolytic agent, and recent advances now allow genetic manipulation of this virus to improve cancer killing and safety. This study was designed to investigate the effectiveness of a genetically engineered NDV in the treatment of peritoneally disseminated gastric carcinoma. NDV mutant virus containing a modified F cleavage site and insertion of enhanced green fluorescent protein (GFP), NDV(F3aa)-GFP, was tested in vitro against human gastric cancer cells by standard cytotoxicity at different multiplicities of infection. To test NDV(F3aa)-GFP in vivo in a peritoneal carcinomatosis gastric tumor model, MKN-74 human gastric cancer cells were injected intraperitoneally (IP) in severe combined immunodeficient mice. Mice were treated with NDV(F3aa)-GFP either once or multiple times after tumor challenge. Effective killing of MKN-74 cells by NDV(F3aa)-GFP was found in vitro. This cancer killing was dose-related and correlated with viral replication. GFP expression was a good marker of infection. The virus was also effective as an antitumor therapy in a peritoneal cancer model that simulates clinical disease. Half the animals treated with virus had no evidence of disease. Genetically engineered NDV [NDV(F3aa)-GFP] administered IP is an effective antitumor therapy against peritoneal carcinomatosis from human gastric cancer in a xenograft model, without significant toxicity. These data provide further rationale for clinical trials involving NDV for peritoneal carcinomatosis from gastric cancer.