Cadmium (Cd) and arsenic (As) are two highly toxic heavy metals and metalloids that coexist in many situations posing severe threats to plants. Our investigation was conducted to explore the different regulatory mechanisms of ryegrass (Lolium perenne L.) responding to individual and combined Cd and As stresses in hydroponics. Results showed that the ryegrass well-growth phenotype was affected by Cd stress of 10mg·L-1. However, As of 10mg·L-1 caused rapid water loss, proline surge, and chlorosis in shoots, suggesting that ryegrass was highly sensitive to As. Transcriptomic analysis revealed that the transcription factor LpIRO2 mediated the upregulation of ZIP1 and YSL6 that played an important role in Cd tolerance. We found that the presence of As caused the overexpression of LpSWT12, a process potentially regulated by bHLH14, to mitigate hyperosmolarity. Indoleacetic acid (IAA) and abscisic acid (ABA) contents and expression of their signaling-related genes were significantly affected by As stress rather than Cd. We predict a regulatory network to illustrate the interaction between transporters, transcription factors, and signaling transduction, and explain the antagonism of Cd and As toxicity. This present work provides a research basis for plant protection from Cd and As pollution. Environmental implicationCd and As are two highly toxic heavy metals and metalloids that coexist in many situations resulting in plant uptake, and then threaten humans through the food chain. Ryegrass is a popular cool-season forage, yet its comprehensively responsive mechanisms under individual and combined Cd and As stresses are poorly studied. Herein, physiological and transcriptomic analysis reveals that ryegrass exhibits efficient adaptation to Cd, and high sensitivity to As, which are associated with their respective responsive transporters and hormone signaling mediated by transcription factors. Our results provide a theoretical basis for alleviating Cd and As co-contamination of plants.