Beneficial bacteria are becoming ever more popular gene delivery method for hypoxia-tumor targeting in vivo. In this study we investigated the therapeutic effect of new recombinant Bifidobacterium breve strain expressing interleukin (IL)-24 gene (B. breve-IL24) on head and neck tumor xenograft in mice. Briefly, B. breve transformants were obtained through electro-transformation. Bacteria-tumor-targeting ability were analyzed in vivo over different time points (1, 3 and 7 days post-bacteria injection). Furthermore, the therapeutic effect of bacteria on tumor cells in vivo were analyzed as follows: 30 Balb/c nude mice bearing subcutaneous tumor were randomly divided in three groups (Drug group, green fluorescent protein (GFP) group and Saline group). The therapy lasted for 2 weeks and included B. breve-IL24 administration via tail vein for Drug group, B. breve-GFP for GFP group and phosphate buffered saline for Saline group. The tumor growth was monitored using standard caliper technique, while the apoptosis induction in vivo was analyzed by Real-time Positron Emission Tomography/Computed Tomography (PET/CT) imaging ([18F]-ML-10 tracer). At the end of the experiment, tumor tissues were collected and analyzed by western blotting. Briefly, our results suggested that our new recombinant bacterium has the capability of targeting tumor tissue in vivo. As for the therapeutic effect, our new strain has revealed to be a promising therapeutic approach against tumor growth in vivo. Briefly, higher tumor growth inhibition and higher tumor cell apoptosis induction were observed in Drug group compared with the GFP and Saline groups. To conclude, a new recombinant strain B. breve-IL24 offers a novel, safe and clinically acceptable therapeutic approach for tumor therapy in vivo.