A monoclonal antibody targeting human epidermal growth factor receptor-2 (HER2), trastuzumab has become a standard treatment for HER2-positive breast cancer. Recent advancements in antibody engineering have enabled the efficient generation of the trastuzumab single-chain variable fragment (scFv). In this study, we genetically engineered Bifidobacterium, a bacterial strain shown to accumulate safely and selectively in hypoxic tumor sites by intravenous (iv) injection, to express and secrete the trastuzumab scFv. The recombinant scFv bound to cell surface HER2 and inhibited in vitro growth of HER2-positive human cancer cells. Moreover, iv-injected recombinant bacteria specifically localized and secreted trastuzumab scFv in xenografted human HER2-positive tumors and consequently inhibited tumor growth. The development and results of this novel in situ delivery and production system for trastuzumab scFv with Bifidobacterium represents a promising avenue for future application in cancer treatment. (C) 2017 Elsevier Inc. All rights reserved.