Biochar, as an amendment to enhance phytoremediation of heavy metal contamination, can mediate reactive oxygen species (ROS) generation. However, the role of biochar-mediated ROS (BMR) during soil-plant phytoremediation remains inadequately understood. In this study, a combination of pot experiments, chemical extraction, and partial least squares path modeling (PLS-PM) was employed to investigate BMR dynamics and their influence on chromium (Cr) accumulation and detoxification in plants. Biochar addition promoted Cr removal efficiency and decreased ROS concentrations in soil, notably reaching the largest removal efficiency of 80.60 % and the lowest ROS concentration of 37.53 μmol/kg in BC-3 group at 90d. Decreased ROS concentrations in soil facilitated the plant absorbing water-soluble Cr (VI), adsorbed Cr (VI), and chromate-precipitated Cr (VI) in soil, and enhanced Cr accumulation in metabolically inactive compartments (cell walls and vacuoles). When biochar was added at concentrations of 2 % and 3 % (w/w), ROS concentrations in plant tissues decreased to signaling molecule thresholds. This reduction further stimulated antioxidant enzyme activity, promoted the reduction of Cr (VI) within subcellular organelles, and enhanced Cr cell wall fixation and vacuolar compartmentation, ultimately achieving their synergistic integration with Cr detoxification with accumulation. This study provides an in-depth understanding of BMR-related mechanisms during phytoremediation and valuable insights into strategies for enhancing mitigation of variable valence heavy metals in soils.