To know more about the photo-inhibition of photosynthesis under field conditions, its relationship with reactive oxygen species (ROS) in leaves of poplar clone seedlings was studied under osmotic stress conditions. With the increasing duration of osmotic stress and the enhancement of stress intensity, the ROS level, malondialdehyde (MDA) content, and superoxide dismutase (SOD) activity increased, while the activities of catalase (CAT) and ascorbate peroxidase (APX) decreased, resulted in the unbalanced ROS metabolism and intensified photo-inhibition. The inhibition of SOD activity by copper diethyldithiocarbamate or the accelerated generation of O2-. with methyl viologen (MV) could also induce the photo-inhibition. The decrease of the ability of poplar clone seedlings in scavenging H2O2 under osmotic conditions limited the dissipation of excess light energy and the role of photosynthetic apparatus in protecting against photo-inhibition through Mehler reaction. Photo-inhibition might be related to the accumulation of ROS.