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BackgroundAcceptance and commitment therapy (ACT) is a promising intervention for improving mental health. However, there is limited evidence on its effectiveness for nurses, particularly in web- and mobile-based intervention forms, in mitigating anxiety and depression symptoms. ObjectiveIn this study, we aimed to examine the effect and underlying psychological mechanisms of a web- and mobile-based ACT intervention on nurses’ anxiety and depression symptoms. MethodsIn this fully decentralized randomized controlled trial, nurses were recruited nationwide across China through advertisements and posters. They were randomly assigned to either the 5-week fully automated intervention or the waiting group. Primary outcomes (anxiety and depression symptoms); secondary outcomes (sleep quality, burnout, and work performance); and mediators (psychological flexibility, cognitive defusion, mindfulness, and values) were assessed using the Wenjuanxing platform. Data collectors were blinded to the group assignments throughout the study period. ResultsA total of 145 nurses with anxiety or depression symptoms were randomly assigned to either the intervention group (n=72, 49.7%) or the control group (n=73, 50.3%); 97.2% (n=141) were female. During the study, 36 (24.8%) nurses were lost to follow-up, and 53 (73.6%) completed the entire intervention. Nurses in the intervention group showed significant improvement in anxiety (d=0.67, 95% CI 0.33-1.00) and depression symptoms (d=0.58, 95% CI 0.25-0.91), and the effects were sustained for 3 months after the intervention (anxiety: d=0.55, 95% CI 0.22-0.89; depression: d=0.66, 95% CI 0.33-1.00). Changes in psychological flexibility, cognitive defusion, and values mediated the effect of the intervention on anxiety and depression symptoms, while mindfulness did not have a mediating effect. ConclusionsThe web- and mobile-based ACT intervention used in this study significantly improved nurses’ anxiety and depression symptoms by improving psychological flexibility, cognitive defusion, and values. The results provide new ideas for hospital administrators to prevent and intervene in nurses’ psychological issues. Trial RegistrationChinese Clinical Trial Register ChiCTR2200059218; https://tinyurl.com/4mb4t5y9