Abstract: Objective: To construct the prokaryotic expression vector pNZ44-ssEndostatin which contains secretable form of mouse endostatin(ssEndostatin) and to express the Endostatin protein in bifidobacterium. Methods: We used primer design software Oligo 6.0 to design appropriate primers. The ssEndostatin gene was amplified from the plasmid pcDNA3.1-Egr1-ssEndostatin by PCR and was then sequenced. When the sequencing results confirmed that the acquired gene was indeed correct, the ssEndostatin gene was ligated to the expression vector pNZ44 to construct the recombinant plasmid pNZ44-ssEndostatin. The vector was electrotransformed into bifidobacterium and the Endostatin protein expression was detected by ELISA. Results: The sequence of ssEndostatin gene acquired by PCR was in concordance with publicly available data,indicating that the ssEndostatin gene was correctly cloned. The recombinant plasmid pNZ44-ssEndostatin was successfully constructed and transformed into bifidobacterium, as shown by colony PCR. The Endostatin protein was expressed inside bifidobacterium, and the quantity was increased by hypoxia. The expression was higher in bifidobacterium cultured for 48hours than in those cultured for 20 hours. Conculsion: The recombinant plasmid pNZ44-ssEndostatin containing Endo-statin with the secretion signal sequence was constructed successfully. The endostatin protein was expressed after the transformation of recombinant plasmid pNZ44-ssEndostatin into bifidobacterium. Bifidobacterium may be used as a tool for endostatin gene therapy.