Alternative splicing and DNA damage exhibit cross-regulation, with not only DNA damage inducing changes in alternative splicing, but alternative splicing itself possibly modulating the DNA damage response (DDR). Sirt1, a prominent anti-aging player, plays pivotal roles in the DDR. However, few studies have examined alternative splicing with DNA damage in neural stem cells (NSCs) and, in essence, nothing is known about whether SIRT1 regulates alternative splicing. Hence, we investigated the potential involvement of Sirt1-mediated alternative splicing in the NSC DDR. Genome-wide alternative splicing profiling was performed upon DNA damage induction and SIRT1 deletion. DNA damage caused genome-wide changes in alternative splicing in adult NSCs and Sirt1 deficiency dramatically altered DDR-related alternative splicing. In particular, extensive alternative splicing changes in DDR-related processes such as cell cycle control and DNA damage repair were observed; these processes were dramatically influenced by Sirt1 deficiency. Phenotypically, Sirt1 deficiency altered the proliferation and DNA repair of adult NSCs, possibly by regulating alternative splicing. SIRT1 helps to regulate alternative splicing, which itself affects the DDR of NSCs. Our findings provide novel insight into the mechanisms underlying the DDR in stem cells.