Mechanoreceptive hair cells are essential for hearing and balance in vertebrates. They have an ability of spontaneous regeneration after damage in lower vertebrate but not in mammals. The lateral line hair cells in fish share structural, functional, developmental and regenerative similarities with those in the inner ear. In order to understand the mechanism of hair cell regeneration and the role of key functional genes,eya1andsix1bduring this process, zebrafish lateral line neuromast hair cells were taken as the study subject in this report. We destroyed neuromast hair cells by treating zebrafish larvae of 5 days postfertilization in 400 μmol/L neomycin for 1 hour. Both the control and treatment groups were observed at 2, 4, 8, 12 and 24 hours post treatment (hpt) via methods of histological sections and in situ hybridization (ISH). It was found that, in control group, hair cells and supporting cells with regular shape were distributed ordinarily in top and lower respectively as two layers; however, the number of the cell in the top layer was significantly decreased during 2-4 hpt. During 8-12 hpt, the boundary between the top and lower layer was indistinct, and some supporting cells were elongated and separated from basement membrane. By 24 hpt, the number, shape, distribution of hair cells and supporting cells in some neuromasts were nearly recovered to the level of control samples. ISH results showed that both eya1andsix1b were expressed in the whole neuromast of the zebrafish larvae in control group. The expression levels of eya1andsix1b in hair cells, but not supporting cells, were significantly reduced during 2-4 hpt. The levels started increasing at 8 hpt, and gradually reached full recovery or even exceeded the level in the control group during 12-24 hpt. Our results indicate that, both the key functional genes eya1andsix1b are reactivated during the hair cell regeneration process in zebrafish lateral line neuromasts and the supporting cells may be involved in hair cell regeneration.