Background: Radiotherapy is a standard adjuvant therapy in patients with progressive rectal cancer, but many patients are resistant to radiotherapy, leading to poor prognosis. Our study identified microRNA-652 (miR-652) value on radiotherapy response and outcome in rectal cancer patients.Methods: miR-652 expression was determined by qPCR in primary rectal cancer from 48 patients with and 53 patients without radiotherapy. The association of miR-652 with biological factors and the prognosis was examined. The biological function of miR-652 was identified through TCGA and GEPIA database searches. Two human colon cancer cell lines (HCT116 p53(+/+) and p53(-/-)) were used for in vitro study. The molecular interactions of miR-652 and tumor suppressor genes were studied through a computational approach.Results: In RT patients, miR-652 expression was significantly decreased in cancers when compared to non-radiotherapy cases (P = 0.002). High miR-652 expression in non-RT patients was with increased apoptosis marker (P = 0.036), ATM (P = 0.010), and DNp73 expression (P = 0.009). High miR-652 expression was related to worse disease-free survival of non-radiotherapy patients, independent of gender, age, tumor stage, and differentiation (P = 0.028; HR = 7.398, 95% CI 0.217-3.786). The biological functional analysis further identified the prognostic value and potential relationship of miR-652 with apoptosis in rectal cancer. miR-652 expression in cancers was negatively related to WRAP53 expression (P = 0.022). After miR-652 inhibition, the estimation of reactive oxygen species, caspase activity, and apoptosis in HCT116 p53(+/+ )cells was significantly increased compared with HCT116 p53(-/-) cells after radiation. The results of the molecular docking analysis show that the miR652-CTNNBL1 and miR652-TP53 were highly stable.Conclusion: Our findings suggest the potential value of miR-652 expression as a marker for the prediction of radiation response and clinical outcome in rectal cancer patients.