Latcripin-13 domain, isolated from the transcriptome of Lentinula edodes C91-3, contains a regulator of chromosome condensation (RCC1) domain/β-lactamase-inhibitor protein II (BLIP-II) and a plant homeodomain (PHD). Latcripin-13 domain has been shown to have antitumor effects. However, the underlying molecular pharmacology is largely unknown. We report here that Latcripin-13 domain induced cell cycle arrest in the G1 phase and caused the apoptosis of human lung carcinoma A549 cells via the GSK3β-cyclin D1 and caspase-8/NF-κB signaling pathways. Western blot analysis showed that Latcripin-13 domain decreased cyclin D1 and cyclin-dependent kinase 4 (CDK4), while it increased the ratio of GSK3β/phosphorylated GSK3β. Importantly, Latcripin-13 domain induced nuclear fragmentation and chromatin condensation in the A549 cells. In addition, treatment of the A549 cells with Latcripin-13 domain resulted in the loss of mitochondrial membrane potential, accompanied by an increase in the Bax/Bcl-2 ratio and activation of caspase-3, -8, and -9. Intriguingly, western blot analysis revealed that NF-κB was significantly downregulated by Latcripin-13 domain. These results demonstrated that Latcripin-13 domain induced apoptosis and cell cycle arrest at G1 phase in the A549 cells, providing a mechanism for the antitumor effects of Latcripin-13 domain.