Tumor stemness maintenance and endoplasmic reticulum (ER) stress response have been strongly correlated with the progression of lung cancer (LC). Nevertheless, the role of long non-coding RNAs (lncRNAs) in these processes remains incompletely understood. We screened LC-associated lncRNAs from the GEO database and validated the expression of TBX5-AS1 in clinical samples. Functional experiments were conducted to assess the biological effects of TBX5-AS1, and western blot was used to detect ER stress marker proteins. The interaction mechanism of the TBX5-AS1/miR-494–3p/ATF6 axis was elucidated through dual-luciferase reporter assays, RNA immunoprecipitation (RIP), and pull-down experiments. Rescue experiments and a nude mouse xenograft model were employed to validate the functional outcomes. TBX5-AS1 was significantly downregulated in LC tissues and cell lines, and its low expression was associated with advanced tumor stages and poor patient prognosis. Overexpression of TBX5-AS1 markedly suppressed LC cell proliferation, migration, invasion, and self-renewal while promoting the activation of the ER stress pathway. Mechanistically, TBX5-AS1 competitively binds to miR-494–3p, thereby relieving its transcriptional repression of Activating transcription factor 6 (ATF6). Rescue experiments demonstrated that miR-494–3p overexpression reversed the regulatory effects of TBX5-AS1 on tumor malignant phenotype and ER stress. In vivo experiments further confirmed that TBX5-AS1 overexpression significantly inhibited tumor growth, accompanied by upregulation of ATF6 and ER stress-related proteins. TBX5-AS1 functioned as a tumor-suppressive lncRNA by activating ER stress signaling through the miR-494–3p/ATF6 axis, thereby inhibiting LC growth and tumor stemness.