Background: Actin-like protein 8 (ACTL8) is a member of the CTA family, and it is expressed in various types of cancer, including glioblastoma and breast cancer. However, whether ACTL8 is involved in the development of lung adenocarcinoma (LUAD) remains unknown. Here, we try to demonstrate the role of ACTL8 in human LUAD A549 cells. Methods: First, the high expression of ACTL8 was observed in patients with LUAD via immunohistochemistry (IHC) staining. Second, cell proliferation was significantly inhibited in ACTL8 knockdown A549 cells. Third, a global gene expression analysis was performed to discover the potential genes and signal pathways modulated by ACTL8 in A549 cells. Results: A total of 504 differentially expressed genes (DEGs) (146 up-regulated, and 358 clown-regulated) were found in the ACTL8 knockdown A549 cells compared with the mock-transfected cells. Ingenuity pathway analysis (IPA) revealed that canonical pathways such as cyclins and cell cycle regulation and estrogen-mediated S-phase entry were significantly inhibited, while pathways such as cell cycle: G2/M DNA damage checkpoint regulation and HMGB1 signaling were significantly activated by ACTL8 knockdown. Disease and functions enrichment analysis revealed that processes associated with "cell death" and "apoptosis" were significantly activated. Upstream regulator analysis showed that NUPR1 was the most activated, while CSF2 was the most inhibited. Lastly, a qRT-PCR and Western blot analysis further confirmed that the expression levels of FOXM1, STMN1, PLK1, and BIRC5 were markedly reduced in ACTL8 knockdown of A549 cells. Conclusions: In summary, these results suggest that a knockdown of ACTL8 inhibits cell proliferation in human LUAD A549 cells by regulating FOXM1, STMN1, PLK1, and BIRC5.