Abstract: Background　Early-stage lung adenocarcinoma often presents as ground-glass nodules on CT imaging and exhibits distinct biological behaviors, thus representing a major focus in the field of early detection and treatment of lung cancer. Although β Ⅲ-tubulin (TUBB3) is aberrantly expressed in various malignancies, its role in the progression of early-stage lung adenocarcinoma remains poorly understood. Objective　To investigate the expression of TUBB3 across different pathological stages of lung adenocarcinoma and in lung adenocarcinoma cell lines, and to investigate its impacts on the biological behaviors of lung adenocarcinoma cell lines.Methods　Expression and survival analyses of TUBB3 were conducted utilizing data extracted from The Cancer Genome Atlas (TCGA) database. A total of 64 lung adenocarcinoma tissues and paired adjacent samples, including atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA), and invasive adenocarcinoma (IAC), were collected between August 2021 and December 2023 from PLA general hospital. Immunofluorescence staining was performed to detect TUBB3 protein expression. Western blot and quantitative real-time PCR were used to detect TUBB3 expression in lung adenocarcinoma cell lines. Stable TUBB3-knockdown A549 cell strains and corresponding controls were constructed via lentiviral transduction. Cell proliferation, migration, invasion, apoptosis, and adhesion abilities were assessed using CCK-8 assay, wound healing assay, Transwell invasion assay, flow cytometry, and adhesion assay, respectively. Lung cancer-on-achip models were established to further evaluate the impacts of TUBB3 on cell proliferation, migration, invasion, apoptosis, and adhesion abilities. Nude mouse subcutaneous xenograft models were constructed to assess tumorigenicity in vivo.Results　TUBB3 expression was significantly upregulated in lung adenocarcinoma tissues (P＜0.001) and was associated with poor prognosis (P＜ 0.05). TUBB3 protein expression was significantly higher in IAC and MIA compared with AAH and AIS (P＜0.001), and higher in AAH and AIS than in adjacent normal tissues (P＜0.001). Elevated TUBB3 expression was also observed in A549 and H292 cell lines (P＜0.01). TUBB3 knockdown markedly suppressed proliferation (P＜0.001), migration (P＜0.001), and invasion (P＜0.01) abilities of A549 cell strains, while enhancing cell adhesion ability (P＜0.05), with no significant impact on apoptosis (P＞0.05). In lung cancer-on-a-chip models, TUBB3 knockdown reduced cell proliferation (P＜0.001) and migration abilities(P＜0.05), decreased epithelial-mesenchymal transition related protein N-cadherin expression (P<0.001), and increased ZO-1 (P＜0.01) and E-cadherin (P ＜0.001) expressions, with no significant effect on apoptosis (P＞0.05). Furthermore, TUBB3 knockdown significantly attenuated the tumorigenic capacity of A549 cell strains in nude mice (P＜0.01). Conclusion　TUBB3 is significantly upregulated in lung adenocarcinoma, with expression progressively increasing during early-stage lung adenocarcinoma progression. Functional analyses demonstrate that TUBB3 knockdown downregulate proliferation, migration, invasion, and tumorigenicity abilities, while enhancing cell adhesion in A549 cell lines.