Objective To investigate the effects and regulatory mechanisms of long non-coding RNA (lncRNA) TUC338 on the proliferation, migration and invasion and epidermal growth factor receptor (EGFR)/phosphoinositide 3 kinase (PI3K)/serine/threonine protein kinase (AKT) of non-small cell lung cancer cells. Methods From July 2019 to June 2021, lung cancer tissue and paracancerous tissue specimens resected from 37 patients with NSCLC by thoracic surgery in Suining Central Hospital were collected. RT-qPCR was used to detect the expression of lncRNA TUC338 in tissue specimens and NSCLC cells. NCI-H157 cells were intervened, and si-TUC338, si-NC, pcDNA-TUC338, pcDNA-NC plasmids were transfected into cells. EGFR and PI3K dual inhibitor MTX-211 inhibitor (MTX-211). pcDNA-TUC338 and MTX-211 were co-treated Cells. CCK-8 assay was used to detect cell proliferation. Transwell chamber assay was used to detect cell migration and invasion. Western blot was used to detect cyclin D1 (cyclin D1), matrix metalloproteinase 2 (MMP-2), MMP-9 protein and EGFR. The phosphoinositide 3 kinase (PI3K), serine/threonine protein kinase (AKT) related protein expression were detected respectively. Results LncRNA TUC338 was highly expressed in lung cancer tissues and cells. Inhibiting the expression of lncRNA TUC338 could significantly inhibit the proliferation, migration and invasion of NCI-H157 cells and the activation of EGFR/PI3K/AKT pathway (P<0.05). Over-expression of lncRNA TUC338 could promote NCI-H157 cells proliferation, migration and invasion and EGFR/PI3K/AKT activation (P<0.05).Conclusion LncRNA TUC338 is highly expressed in lung cancer, which may promote the proliferation, migration and invasion of lung cancer NCI-H157 cells by activating the EGFR/PI3K/AKT signaling pathway.