It has been known that ABA INSENSITIVE 5 (ABI5) plays a vital role in regulating seed germination. In the present experiment, we showed that 3-amino-1,2,4-triazole (3-AT) inhibits seed germination of the loss-of-function mutant abi5-1, but promotes seed germination of the ABI5-overexpression transgenic line, and that ABI5 affects reactive oxygen species (ROS) homeostasis. RNA-sequencing analysis indicates that the transcription of the genes involved in ROS metabolic process or genes responsive to ROS stress is impaired in abi5-1 seeds. Together, all the findings suggest that ABI5 regulates seed germination at least partly by affecting ROS homeostasis. Overall design: For RNA sequencing (RNA-Seq) analysis, total RNA was isolated from imbibed seeds of wild type and abi5-1 plants. The control sample is the wild type. For each sample, at least three biological replicates were sequenced.