Abstract： Objective To investigate the enhancing effect of ultrasound microbubbles on transfection of recombinant adeno-associated virus (rAAV) mediated green fluorecent protein (EGFP) gene into retinal ganglion cells (RGC) in vivo. Methods A total of 40 adult Sprague-Dawley (SD) rats were divided into four groups randomly (group A, B, C, D) with 10 rats in each. Group A was the normal control, in which the rats underwent intravitreal injection with 5 μl phosphate buffered solution. The rats in group B underwent intravitreal injection with 5 μl recombinant adeno-associated virus encoding EGFP gene (rAAV2-EGFP). The rats in group C underwent ultrasound irradiation on eyes right after intravitreal injection with 5μl rAAV2-EGFP; The ultrasound irradiation was performed on the rats in group D right after intravitreal injection with the mixture solution of microbubbles and rAAV2-EGFP ultrasound. After 21 days, RGC were labeled retogradely with fluogold. Seven days after labeling, the retinal flatmounts and frozen sections were made from five rats in each group. Expression of EGFP reporter gene was observed by laser scanning confocal microscope and evaluated via average optical intensity (AOD) and RGC transfection rate. Labeled RGC were counted to evaluate the adverse effects. Results Green fluorescence can be observed exactly in labeled RGC in B,C, and D groups. The AOD and transfection rate in group D was (95.02 ± 7. 25)% and (20. 10±0. 74)% , respectively; which were higher than those in group B and C (F=25. 970,25. 799;P<0.01). The difference of the number of RGC among the four groups was not significant(F= 0. 877, P>0. 05). Conclusion Under the condition of low frequency and with certain energy, ultrasound-mediated microbubble destruction can effectively and safely enhance rAAV delivery to RGC in rats.