Objective:To investigate the efficiency of transducing rat bone marrow mesenchymal stem cells(BMSCs) with enhanced green fluorescent proteins(eGFP) gene in vitro by a chimeric adenovirus serotype 5 vector Ad5/F35 which carries an adenovirus serotype 35 fiber and observe the longevity of transgene expression in transduced rat BMSCs. Methods:Rat BMSCs was isolated and proliferated by adhesive culture; transduced rat BMSCs of passage 2 with eGFP gene using Ad5/F35 carrying an eGFP gene(Ad5/F35-eGFP) at a multiplicity of infection(MOI) of 1,10,100 and 1000 respectively; tested the transduction efficiency and expression level of eGFP gene in rat BMSCs by fluorescent inverted microscope and flow cytometer(FCM); studied the basic biological characteristic of transduced rat BMSCs by observing their growing state; tested the eGFP expression in rat BMSCs at day 2,7,14,21 and 30 respectively after transduction at a MOI of 100 by FCM. Results:A positive dose-response relationship was observed between the transduction efficiency and expression of eGFP gene and the Ad5/F35-eGFP concentrations ranging from 1~1000 MOI. Approximately 90% of the rat BMSCs can be transduced by Ad5/F35-eGFP at a MOI of 100,and the transgene expression can be detected for a month. The virus which infects rat BMSCs at a MOI of 1,10 and 100 can not impair the vitality and proliferative capability of transduced rat BMSCs. Conclusion:Ad5/F35-eGFP can transduce rat BMSCs with eGFP gene efficiently in vitro,the transgene expression in rat BMSCs can last a month. These data suggest that the Ad5/F35 is an effective vector of BMSCs for gene transfer and expression and it will be a powerful tool for gene therapy and cell therapy based on BMSCs.