Aldo-keto reductase 1C3 (AKR1C3), one member of the aldo-keto reductase superfamily, is involved in a variety of cancers. Recently, AKR1C3 has been demonstrated to be related with the doxorubicin (DOX) resistance in human breast cancer. Here, we attempted to explore the resistance mechanism mediated by AKR1C3. First, one DOX resistant breast cancer cell line MCF-7/DOX was successfully established and an increased level of AKR1C3 was observed in the MCF-7/DOX cells compared to the parental MCF-7 cells. To investigate the contribution of AKR1C3 in the DOX resistance, we further established an AKR1C3 overexpression cell line, referred to MCF-7/AKR1C3. In the MCF-7/AKR1C3 cells, the DOX induced cytotoxicity, detected by CCK-8 cell viability assay and DAPI staining, was greatly reduced (3.2-fold increase in the IC₅₀ value). Interestingly, a loss of tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) was observed when AKR1C3 was overexpressed. Secondary to the PTEN loss, the activated Akt also markedly increased. In addition, the AKR1C3 mediated DOX resistance can be conquered by the Akt inhibitor (LY294002). Furthermore, we found that the expression levels of AKR1C3 and PTEN had a negative relationship in the human breast tumor tissues (the standard correlation coefficient=−0.71; P=0.048). In conclusion, our data suggested that the AKR1C3 mediated DOX resistance might be resulted from the activation of anti-apoptosis PTEN/Akt pathway via PTEN loss. AKR1C3 may present a potential therapeutic target in addressing DOX resistance in breast cancer.