The emergence of drug resistance is still a daunting challenge for the effective therapy of cancer patients. miRNAs have been elucidated as an important regulator in chemoresistance of anti-cancer drugs. miR-381 is found to exert tumor-suppressive effect in breast cancer. However, its role in modulating the sensitivity of doxorubicin (DOX) remains unknown. In this study, we found that miR-381 expression was down-regulated in DOX-resistant breast cancer cells. miR-381 overexpression increased DOX sensitivity and enhanced DOX-induced apoptosis in breast cancer cells. Moreover, miR-381 could directly target FYN to suppress its expression. Additionally, FYN knockdown displayed similar effect on DOX sensitivity as miR-381 up-regulation. Furthermore, FYN overexpression partly reversed miR-381-induced sensitivity to DOX. Finally, enforced expression of miR-381 also improved DOX sensitivity of breast cancer cells in vivo. In summary, miR-381 inactivated MAPK signaling by down-regulating FYN, thereby promoting the chemosensitization of breast cancer cells to DOX. Therefore, miR-381/FYN/MAPK pathway may be applied as a novel target to overcome DOX resistance in breast cancer patients.