Objective To assess the changes of humidity and ammonia concentration in rat and mouse individually ventilated cages (IVC) based on macroenvironmental humidity and air ventilation changes. Methods Three kinds of rat and mouse IVC in barrier facilities were set as research objective. The changes of micronvironmental humidity and ammonia concentration at 40 times/h and 60 times/h air changes were detected continuously for a 7-days-cycle relative to low (40%), moderate (50%), and high (60%) macroenvironmental humidity. Results Mouse and rat IVC with 40 times/h air changes under low macroenvironmental humidity condition, mouse IVC with 40 times/h and rat IVC with 60 times/h air changes under moderate macroenvironmental humidity condition, mouse IVC with 60 times/h air changes under high macroenvironmental humidity condition, basically meet the GB14925-2010 requirements. While under macroenvironmental high humidity condition, the microenvironments of rat and mouse IVC with 60 times/h air changes could not satisfy the requirements. Conclusions The environmental humidity and ventilation frequency are the key index of IVC microenvironment. Only on the basis of external environment conditions to set up reasonable IVC ventilation frequency in order to better maintain the IVC microenvironment so that to achieve the goal of effective management.