Long non-coding (lnc)RNAs have been found to play a crucial role in tumor progression. The present study aimed to investigate the association between lncRNA RASSF8-AS1 and laryngeal squamous cell carcinoma (LSCC) and the underlying mechanisms. Reverse transcription-quantitative PCR was used to measure the mRNA expression level of RASSF8-AS1, microRNA(miR)-664b-3p and transducin-like enhancer of split 1 (TLE1) in LSCC. The associations between RASSF8-AS1 and miR-664b-3p, and between miR-664b-3p and TLE1 were investigated using a dual luciferase reporter assay, while the former was further verified using an RNA immunoprecipitation (RIP) assay. The association between RASSF8-AS1 and miR-664b-3p on cell biological functions was investigatedin vitrousing MTS, colony formation and Transwell assays. The RASSF8-AS1 mRNA expression level was decreased in LSCC cell lines and carcinoma tissues, while overexpression of RASSF8-AS1 reduced the migration, invasion and proliferation abilities of LSCC cells. Furthermore, luciferase and RIP assays confirmed that RASSF8-AS1 was a competitive endogenous (ce)RNA by sponging miR-664b-3p to activate TLE1. miR-664b-3p was negatively modulated by RASSF8-AS1; however, TLE1 was positively regulated by RASSF8-AS1. Functionally, RASSF8-AS1 acted as a ceRNA to upregulate TLE1 by sponging miR-664b-3p. In conclusion, the RASSF8-AS1/miR-664b-3p/TLE1 axis acts by suppressing LSCC progression and may provide a novel insight for the molecular mechanism of LSCC.