Bifidobacterium longum (B. longum) as a delivery system for endostatin was shown to have definite antitumor effects. Moreover, it was found that the enrichment of selenium was able to enhance the immunity of mice. In order to further evaluate the safety and efficacy of B. longum carrying pBV22210-endostatin (B. longum-En) enriched with selenium (Se-B. longum-En), we determined the biochemical characteristics of Se-B. longum-En. We then investigated its effect on macrophage activity, as well as its inhibitory effect on the multiplication of pathogenic bacteria in vitro and the antitumor effects on murine hepatic (H22) tumor-bearing mice. The results showed that Se-B. longum-En exhibited similar biochemical characteristics to that of wild-type B. longum, i.e., Se-B. longum-En strongly enhanced macrophage phagocytosis in rats and inhibited the growth of pathogenic bacteria. In addition, Se-B. longum-En showed a definite inhibitory effect of tumor growth when H22 tumor-bearing mice were fed through oral or tail vein delivery. These results suggested that Se-B. longum is able to retain the advantages of wild-type B. longum and be used as a novel gene delivery system for liver cancer gene therapy.