Background Non-small cell lung cancer (NSCLC) is the leading cause of cancer-associated mortality worldwide of which lung adenocarcinoma (LUAD) is the most common. The identification of oncogenes and effective drug targets is the key to individualized LUAD treatment. Actin-like protein 8 (ACTL8), a member of the cancer/testis antigen family, is associated with tumor growth and patient prognosis in various types of cancer. However, whether ACTL8 is involved in the development of LUAD remains unknown. The aim of the present study was to demonstrate the role of ACTL8 in human LUAD cells. Methods The expression of ACTL8 in LUAD tissues and cell lines was assessed using immunohistochemistry and western blotting. Additionally, plasmids expressing ACTL8-specific short hairpin RNAs were used to generate lentiviruses which were subsequently used to infect A549 and NCI-H1975 human LUAD cells. Cell proliferation, migration, invasion and apoptosis, as well as cell cycle progression and the expression of protein markers of epithelial to mesenchymal transition were investigated. A549 cell tumor growth in nude mice was also examined. Results The results showed that ACTL8 was highly expressed in A549 and NCI-H1975 LUAD cell lines. Additionally, ACTL8-knockdown inhibited proliferation, colony formation, cell cycle progression, migration and invasion, and increased apoptosis in both cell lines. Furthermore, in vivo experiments in nude mice revealed that ACTL8-knockdown inhibited A549 cell tumor growth. Conclusion These results suggest that ACTL8 serves an oncogenic role in human LUAD cells, and that ACTL8 may represent a potential therapeutic target for LUAD. Key points Our results suggest that ACTL8 serves an oncogenic role in human LUAD cells, and that ACTL8 may represent a potential therapeutic target for LUAD.