Seed germination is comprehensively regulated by multiple intrinsic and extrinsic factors, and reactive oxygen species (ROS) are relatively new among these factors. However, the role and underlying mechanisms of ROS in germination regulation remain largely unknown. In this study, we initially found that cold stratification could promote germination and respiration of Hedysarum scoparium seeds, especially at low temperature. We then noted that a ROS environment change induced by hydrogen peroxide (H2O2) or methylviologen (MV) could similarly promote seed germination. On the other hand, the ROS scavenger N-acetyl-L-cysteine (NAC) suppressed germination of cold-stratified H. scoparium seeds, indicating a stimulatory role of ROS upon seed germination. An increased accumulation of O2- was detected in embryonic axes of cold-stratified seeds, and stratification-induced ROS generation as well as progressive accumulation of ROS during germination was further confirmed at the cellular level by confocal microscopy. Moreover, protein carbonylation in cold-stratified seeds was enhanced during germination, which was reversed by NAC treatment. Finally, the relationship between ROS and abscisic acid (ABA) or gibberellin (GA) in germination regulation was investigated. ABA treatment significantly inhibited germination and reduced the H2O2 content in both cold-stratified and non-cold-stratified seeds. Furthermore, we found that cold stratification mediates the down-regulation of the ABA content and increase of GA, suggesting an interaction between ROS and ABA/GA. These results in H. scoparium shed new light on the positive role of ROS and their cross-talk between plant hormones in seed germination.