Allelopathy is prevalent in agricultural ecosystems and mediated by plant-derived secondary metabolites (allelochemicals). Allelochemicals are released by donor plants and affect the root growth and development of receptor plants. Allelopathy is responsible for the continuous cropping obstacles in cucumber (Cucumis sativus L.). p-Hydroxybenzoic acid (pHBA), an autotoxin from root exudates of cucumber, has been proposed to be an important allelopathic chemical. However, the molecular mechanism by which pHBA affect root growth and development in cucumber is unknown. Here, we found that pHBA treatment suppressed root growth of cucumber by reducing the meristem activity and cell length. This root growth defect is caused by reduced reactive oxygen species (ROS) accumulation in root tips. After pHBA treatment, the expression levels of several ROS-scavenging-related genes were increased, including peroxidase (POD), catalase (CAT) and metallothionein (MT). Moreover, exogenously application of salicylhydroxamate (SHAM), a peroxidase inhibitor, can partially restore the pHBA treatment induced root growth inhibition. Furthermore, we found that there is natural variation for the inhibitory effect of pHBA on root growth. We also showed that pHBA treatment could maintain higher level of ROS accumulated in the pHBA less sensitive cucumber than that in the pHBA-sensitive cucumber. These results suggest that pHBA inhibits root growth by reducing root tip ROS level in cucumber.