Many animals regenerate lost body parts through several signalling pathways; however, the triggers that initiate regeneration remain unclear. In the present study, we focused on the role of reactive oxygen species (ROS) produced by the NADPH oxidase Dual oxidase (Duox) during cricket leg regeneration. The results showed that ROS levels were upregulated during leg regeneration and decreased by DuoxRNAi. In DuoxRNAi nymphs, wound closure and scab formation were incomplete 2 days post amputation (dpa), and hypertrophy occurred in the distal region of the regenerating legs at 5 dpa. In addition, the hypertrophic phenotype was induced by DuoxARNAi and NADPH oxidase inhibitor treatment. During hypertrophy, haemocytes, including plasmatocytes, oenocytoids, and granulocytes, accumulated. Proliferation of haemocytes in regenerating legs was not increased by DuoxRNAi; however, haemocyte accumulation was regulated by the Spatzle (Spz) family molecules, which are Toll receptor ligands. As the exoskeleton of DuoxRNAi nymphs was thinner than that of the control, excessive haemocyte accumulation can cause hypertrophy in DuoxRNAi nymphs. Thus, Duox-derived ROS are involved in wound healing and haemocyte accumulation through Spz/Toll signalling pathway during leg regeneration in crickets.