Long non-coding RNAs are involved in the tumorigenesis of non-small cell lung cancer (NSCLC). Here we investigated whether LINC00476 affects the proliferation, invasion and migration of NSCLC cells via the SETDB1-activated Wnt/β-catenin pathway. The expression of LINC00476, SETDB1, Wnt1 and β-catenin were determined in NSCLC tumor tissues and the paired adjacent tissues, as well as in NSCLC cell lines and bronchial epithelioid cell lines. Cell proliferation, invasion and migration were determined using cell counting kit-8 assay and transwell assay. The relationship between LINC00476 and SETDB1 was elucidated using RNA pull-down, RNA immunoprecipitation and ubiquitination assays. LINC00476 was found to be significantly downregulated, while SETDB1, Wnt1 and β-catenin were upregulated in NSCLC tumor tissues and cell lines compared to the normal ones. Overexpression of LINC00476 promoted the proliferation, invasion and migration of NSCLC cells, and suppressed tumor growth in the mouse xenograft. Meanwhile, overexpression of LINC00476 induced the degradation of SETDB1 by promoting its ubiquitination. The simultaneous overexpression of LINC00476 and SETDB1 negated the inhibition of LINC00476 overexpression on the proliferation, invasion and migration of NSCLC cells. In conclusion, these findings indicate that LINC00476 acts as a tumor suppressor in NSCLC by downregulating SETDB1, which provides a novel target in the treatment of NSCLC.