During the development of the vertebrate nervous system, 50% of the nerve cells undergo apoptosis shortly after formation. This process is important for sculpting tissue during morphogenesis and removing transiently functional cells that are no longer needed, ensuring the appropriate number of neurons in each region. Dysregulation of neuronal apoptosis can lead to neurodegenerative diseases. However, the molecular events involved in activating and regulating the neuronal apoptosis program are not fully understood. In this study, we identified several RB1 mutations in patients with neurodegenerative diseases. Then, we used a zebrafish model to investigate the role of Rb1 in neuronal apoptosis. We showed that Rb1-deficient mutants exhibit a significant hindbrain neuronal apoptosis, resulting in increased microglia infiltration. We further revealed that the apoptotic neurons in Rb1-deficient zebrafish were post-mitotic neurons, and Rb1 inhibits the apoptosis of these neurons by regulating bcl2/caspase through binding to Kmt5b. Moreover, using this zebrafish mutant, we verified the pathogenicity of the R621S and L819V mutations of human RB1 in neuronal apoptosis. Collectively, our data indicate that the Rb1-Kmt5b-caspase/bcl2 axis is crucial for protecting post-mitotic neurons from apoptosis and provides an explanation for the pathogenesis of clinically relevant mutations.