Abstract The hair cells and their neural innervation in the avian inner ear can regenerate after injury. Identifying the genes involved in the regeneration and neuroplasticity of avian hair cell will enable us to experimentally induce new hair cell production and potentially harness this process for therapeutic replacement of hair cells in mammals and ultimately in humans suffering from sensorineural hearing loss. In this study, we developed a method for suppressing the expression level of genes in avian inner ear by intratympanic injection of shRNA-expressing lentivirus. The intratympanic injection approach is more convenient and presumably of less implication when compared with two existing methods, in which a nano-particles or gelfoam containing a recombinant virus is placed in the middle ear by surgery, or a recombinant virus is directly injected into the inner ear. Thus, we developed an easier method for identifying and characterizing molecules involved in the process of avian hair cell regeneration and re-innervation.