Objective To investigate the effect of the expression level of S100 calcium-binding protein A16 in lung adenocarcinoma (LUAD) tissues on patient prognosis, and the effect of overexpression of S100A16 on the malignant behavior of lung cancer cells. Methods The expression level of S100A16 in lung cancer microarray was detected by immunohistochemistry. According to the expression level of S100A16 in tumor tissues, immunohistochemical scores were performed to statistically analyze the relationship between S100A16 expression and clinicopathological parameters and patient prognosis. The gene expression of S100A16 in lung adenocarcinoma tissues and normal tissues was analyzed by GEPIA database. After S100A16 was knocked down in lung cancer cell line, cell migration and invasion ability were detected by cell scratch assay and Transwell assay, and the proliferation ability of lung cancer cells was detected by Edu assay. Results Immunohistochemical results showed that the expression level of S100A16 was significantly up-regulated in tumour tissues compared to paraneoplastic tissues, and the elevated level of S100A16 expression was associated with lymph node metastasis, tumour stage and distant organ metastasis (P<0.05), but not related to differentiation degree, age, or gender (P>0.05). Survival analysis showed that the higher the level of S100A16 expression in tumour tissue, the worse the prognosis of patients (P<0.001). The vitro experiment showed that inhibition of S100A16 expression suppressed the proliferation and invasive ability of tumour cells (P<0.001). Conclusion The expression level of S100A16 in LADC tumor tissues is elevated, and its expression level is related to clinicopathological parameters. Patients with elevated S100A16 expression tend to have a poorer prognosis. The expression level of S100A16 can regulate the proliferation and invasion of tumor cells.