Objective To investigate the mechanism by which wild-type PTEN gene reversing multi-drug resistance (MDR) in human leukemia K562/ADM cells resistant to adriamycin (ADM). Methods The recombinant adenovirus containing green fluorescent protein and PTEN (Ad-PTEN-GFP)or empty vector (Ad-GFP) was transducted into K562/ADM cells resistant to ADM. The transduction efficiency was assessed by flow cytometry (FCM). Then the cells were treated with different concentrations of ADM, cytarabine (Ara-C) or arsenic trioxide(As₂O₃) 3 days after transduction. The proliferation of K562/ADM cells was examined by MTT assay, the apoptosis rate was assessed by FCM, and the IC₅₀ of different drugs was used to calculate the drug resistance reversal fold (RF), so as to observe the effect of PTEN on reversing MDR of the 3 drugs. PTEN, NF-κB, MDR1, MDR-associated protein (MRP) and apoptosis related genes (Bcl-2, Bcl-xL, Bax) were detected by fluorescence quantitative PCR. PTEN, Akt, p-Akt and NF-κB protein levels were detected by Western blotting analysis. Results The proliferation inhibition rate and apoptosis rate of cells in Ad-PTEN-GFP plus chemotherapeutic groups were significantly higher than those Ad-GFP plus chemotherapeutic groups at 3 days after infection (MOI=200) (P<0.05). PTEN transduction promoted the sensitivity of K562/ADM cells to ADM, Ara-C and As₂O₃, with the RF being 3.80, 2.65 and 2.64 folds, respectively. K562/ADM cells in Ad-PTEN-GFP group had lower p-Akt and NF-κB (P65) protein levels and lower NF-κB, MDR1, Bcl-2 and Bcl-xL mRNA levels, and up-regulated Bax mRNA level compared with those in Ad-GFP group. Conclusion Wild-type PTEN gene may reverse drug resistance via inhibiting Akt pathway and regulating its downstream signaling molecules, such as NF-κB, MDR1, Bcl-2 and Bax.