Background: Lung adenocarcinoma (LUAD) is one of the most prevalent human cancers worldwide. The homeobox-B (HOXB) gene cluster has been reported to contribute to cancer development. Nevertheless, the expression status, clinical significance and biological role of HOXB genes in LUAD remain largely unclear. Methods and results: This study comprehensively investigated the transcriptional levels and prognostic values of the HOXB genes in LUAD based on The Cancer Genome Atlas (TCGA) database. Flow cytometry, CCK-8, and Transwell assays were used for detecting apoptosis, proliferation, and migration, respectively. We discovered that eight members of the HOXB cluster genes (HOXB2, HOXB3, HOXB4, HOXB6, HOXB7, HOXB8, HOXB9, and HOXB13) were dysregulated in LUAD tumor tissues. Increased expression of HOXB3, HOXB6, HOXB7, HOXB8, or HOXB9 was independently associated with unsatisfactory overall survival (OS) in LUAD patients. In addition, a high level of HOXB3 also predicted poor patient relapse-free survival (RFS), suggesting that HOXB3 may play a vital role in the progression of LUAD compared to other members of the HOXB cluster. Additionally, further analysis by TIMER and TISIDB algorithms revealed that HOXB3 was positively correlated with a panel of immune checkpoint molecules (ICMs), tumor-infiltrating lymphocytes (TILs), and tumor immune regulators (TIRs). Gene enrichment analysis based on KEGG showed that HOXB3 was closely associated with multiple tumor-related biological processes and signaling pathways. Functionally, the in vitro experiments revealed that depletion of HOXB3 significantly alleviated the resistance of LUAD cells to apoptosis, and suppressed cell proliferation and migration. Conclusion: Our study suggests that HOXB3 may play an oncogenic role in LUAD and correlate with tumor immunity.