Objective Most of the airborne pathogens of infectious animal experiments in biosafety laboratories can be effectively controlled by barrier apparatus. However,the requirements for biosafety parameters of the rodent cages, which are commonly-used devices, are not clear yet. The purpose of this study is to estimate the risk of pathogen exposure by mimicking infectious animal experiments using open cage or sealed cage for rodents in barrier facility under negative pressure in our laboratory, and to evaluate the risk-controlling level of the hardware conditions and management measures of our facility. Methods E. coli (DH5α, with the kanamycin-resistance gene, Kan⁺) was used as the indicator microorganism. The risk of pathogen exposure of the following operations were tested:mice raised in open cage or sealed cage (without pressure gradient inside and outside), and sealed-cage changing of mice in biosafety cabinet. The quantities of indicator microorganisms inside and outside the facility with negative pressure were monitored to estimate the efficiency of biosafety-control over the facility. Results There was a risk of pathogen exposure when open cage was used in the barrier facility under negative pressure, and the spread of pathogens could be controlled in barrier facility under negative pressure to some extent. There was no indicator microorganism detected when animals were raised in sealed cage without pressure gradient inside and outside, and the risk of pathogen exposure during the procedure of sealed-cage changing in the biosafety cabinet could be effectively controlled. Conclusions The biosafety of animal experiments can be ensured using sealed cage in barrier facility under negative pressure, even without pressure gradient inside and outside of the cage.