Abstract: Objective To construct a novel tuberculosis vaccine candidate LIΔinlB1-Ag85C by knocking out the inlB1 gene of Listeria ivanovii (LI) recombinant strain LI-Ag85C, and study the biological characteristics of the attenuated strain in vitro and in vivo. Methods Targeting plasmid carrying inlB1 upstream and downstream sequences was constructed and electroporated into LI-Ag85C competent cells. Afterward inlB1 gene was knocked out by homologous recombination. Recombinant attenuated strain LIΔinlB1-Ag85C and parental strain LI-Ag85C were tested in growth characteristics, hemolyticability, the adhesion and invasion tendency to HepG2 in vitro and the median lethal dose (LD50) for C57BL/6 mice in vivo. Results Genome sequence of the attenuated tuberculosis vaccine candidate LIΔinlB1-Ag85C was as expected. The attenuated strain and the parental strain showed the similar growth curve in vitro. The adhesion rates of the two strains were 6.66% and 7.46%, respectively, and the invasion rate of them were 0.031% and 0.042% respectively. LIΔinlB1-Ag85C seemed having a lower adhesion and invasion rates to HepG2 cells, however the difference had no significance. The hemolytic ability of recombinant strain was the same as to the parental strain. The LD50 of LIΔinlB1-Ag85C and LI-Ag85C for C57BL/6 mice were 3.2×108 CFU/per mouse and 6.7×107 CFU/per mouse, respectively. LIΔinlB1-Ag85C showed a significantly decrease in animal virulence. Conclusion A novel tuberculosis vaccine candidate LIΔinlB1-Ag85C based on attenuated Listeria ivanovii was successfully constructed with a significant decrease in toxicity.