Zebrafish neuromast is an ideal model for investigating hair cell (HC) death and regeneration following ototoxic insults. HC undergoes rapid and robust replacement in larval zebrafish after neomycin damage. However, the origin of new HCs remains unclear. Our data showed that asymmetric cell division was involved in the process of HC regeneration in zebrafish lateral line neuromasts. Furthermore, a small molecule RRD251, which disrupted the physical interaction between RB and Raf-1 and then blocked the phosphorylation of Rb, could have inhibited the HC regeneration from supporting cell proliferation. Our results indicate that Rb-Raf-1 interaction plays an important role in spontaneous HC regeneration in zebrafish.