Background: Usnic Acid (UA), also known as lichenol, has been reported to have inhibitory effects on a variety of cancer cells, but its specific mechanism remained to be elucidated. Tumor chemotherapy drugs, especially DNA damage chemotherapeutic drugs, target Chromosomal DNA, but their spontaneous and acquired drug resistance are also an urgent problem to be solved. Therefore, drug combination research has become the focus of researchers. Methods: Here, we evaluated the tumor-suppressing molecular mechanism of UA in colorectal cancer cells RKO from the perspective of the ATM-mediated DNA damage signaling pathway through H2O2 simulating DNA damage chemotherapeutic drugs. CCK8 cell proliferation assay was used to determine the inhibition of RKO cells by hydrogen peroxide and UA alone or in combination, and wound healing assay was applied to determine the effect of the drug on cell migration. Results: Transfected cells with miRNA18a-5p mimics and inhibitors, MDC and DCFH-DA staining for the measurement of autophagy and ROS, cell cycle and apoptosis were detected by flow cytometry, expressions of microRNA and mRNA were determined by fluorescence quantitative PCR, and protein by Western blot. Discussion: We found that UA can upregulate ATM via miR-18a to activate the DNA damage signaling pathway and inhibit the proliferation and migration of RKO cells in a concentration-dependent manner. Conclusion: At the same time, DNA damage responses, including cell cycle, autophagy, apoptosis and ROS levels, are also regulated by UA. Therefore, UA combined with DNA damage chemotherapeutic drugs may be an effective treatment for cancer.