Lung cancer treatment remains a challenge for clinical practice and new therapeutic approaches are urgently needed. Loss of functional WEE1 kinase causes DNA replication stress, DNA damage and unscheduled mitotic entry due to elevated CDK activity. The selective WEE1 inhibitor MK-1775 synergize with DNA-damaging agent to inhibit cancer cell growth. Here we report that inhibition of Sirt1 deacetylase through small interfering RNA or selective inhibitor Ex527 greatly enhances MK-1775-induced growth inhibition and apoptosis in human lung cancer cells. We further demonstrate that Sirt1 interacts and deacetylates homologous recombination (HR) repair machinery proteins, including NBS1 and Rad51. Inhibition of Sirt1 impairs HR repair activity, which causes unrepairable damage when combining MK-1775 and Ex527. Meanwhile, combination of MK-1775 and Ex527 induces cooperative antitumor activity in lung cancer xenograft model in vivo. Thus, our study provides a novel therapeutic strategy to optimize MK-1775 treatment efficiency in lung cancers.