This study was performed to investigate the effect of heat-stressed environment on rumen microbial diversity in Holstein cows. Rectal temperature and respiration rate were measured and rumen fluid was collected under normal environment (NE; Temperature humidity index (THI)=64.6) and heat-stressed environment (HE; THI=87.2) from 10 Holstein cows (60±17.7 months, 717±64.4 kg) fed on the basis of dairy feeding management in National Institute of Animal Science. The rumen bacteria diversity was analyzed by using the Illumina HiSeq™ 4000 platform. The rectal temperature and respiratory rate were increased by 1.5ﾟC and 53 breaths/min in HE compared to that in NE, respectively. In this study, HE exposure induced significant changes of ruminal microbe. At phylum level, Fibrobacteres were increased in HE. At genus level, Ruminococcaceae bacterium P7 and YAD3003, Butyrivibrio sp. AE2032, Erysipelotrichaceae bacterium NK3D112, Bifidobacterium pseudolongum, Lachnospiraceae bacterium FE2018, XBB2008, and AC2029, Eubacterium celulosolvens, Clostridium hathewayi, and Butyrivibrio hungatei were decreased in HE, while Choristoneura murinana nucleopolyhedrovirus, Calothrix parasitica, Nostoc sp. KVJ20, Anabaena sp. ATCC 33047, Fibrobacter sp. UWB13 and sp. UWB5, Lachnospiraceae bacterium G41, and Xanthomonas arboricola were increased in HE. In conclusion, HE might have an effect to change the rumen microbial community in Holstein cows.