Efficiently delivering nucleic acid into mammalian cells is essential to overexpress genes for assessing gene functions. Human bone marrow stem cells (hBMSCs) are the most studied tissue-derived stem cells. Adeno-associated viruses (AAVs) have been used to deliver DNA into hBMSCs for various purposes. This study optimized the AAV transduction protocol for high-efficiency DNA delivery into hBMSCs. hBMSCs were infected with different serotypes of AAVs containing green fluorescence (eGFP) reporter driven by the CMV promoter. GFP was monitored in the infected cells. Cells were collected at designated time points after the infection for RT-PCR to assess eGFP mRNA. The results indicated that the order of transduction efficiency of the AAV serotypes was AAV2 > AAV2.7 > AAV6 > AAV6.2 > AAV1 > AAD-DJ. AAV2 could achieve almost 100% transduction at the MOI greater than 100K. Over 90% of cells could be transduced at 20K to 50K MOI. About 80% transduction was seen at MOIs of 10K and 15K. The eGFP expression reached the maximum about15- 25 days post-AAV2 infection. High levels of transcription were still detectable at day 30 post-infection. We conclude that AAV2 and AAV2.7 can efficiently deliver transgene into hBMSCs for sustained expression over one month.