Nowadays, radio-resistance remains a grim problem occurs in patients with breast cancer. This study was aimed to explore the role of microRNA-122-3p (miR-122-3p) on breast cancer cells radio-sensitivity. Human breast cancer cells line MDA-MB-231 was used and transfected with miR-122-3p mimic, inhibitor or control. Transfected cells were exposed to various doses of irradiations, and then the surviving rate, apoptosis, cells migration and invasion were detected by clonogenic survival assay, flow cytometry, and transwell assay respectively. Furthermore, Western blot analysis was used to detect the expression changes of phosphatase and tensin homolog/phosphatidylinositol- 3-kinase/serine/threonine kinas (PTEN/PI3K/AKT) pathway and epithelial-mesenchymal transition (EMT) related proteins in transfected cells after irradiation. The surviving rate of MDA-MB-231 cells was significantly decreased by miR-122-3p overexpression (P < 0.05 or P < 0.01). Apoptosis was induced by miR-122-3p overexpression (P < 0.001) while cells migration and invasion were inhibited by miR-122-3p overexpression (P < 0.001). Besides, miR-122-3p overexpression significantly down-regulated the expressions of p-AKT, AKT, Vimentin and Snail 1 (P < 0.01 or P < 0.001), whereas significantly up-regulated the levels of PTEN and E-Cadherin (P < 0.01). MiR-1223p suppression showed the inversed impacts on the surviving rate, apoptosis, cells migration and invasion, as well as these proteins expressions. MiR-122-3p enhanced the radio-sensitivity of breast cancer cells via controlling of cell apoptosis, migration and invasion. PTEN/PI3K/AKT pathway and ETM related proteins were implicated in this enhancement impacts.