Background and Aims The authors have previously found that the long non-coding RNA FoxP4-AS1 (lncRNA FoxP4-AS1) is abnormally expressed in papillary thyroid carcinoma (PTC) tissue, and its low expression is an independent risk factor for regional lymph node metastasis in PTC. However, its function and action mechanism remain unclear. Therefore, this study was conducted to investigate the expression of lncRNA FoxP4-AS1 in PTC cells, and make a preliminary analysis of its effect on the biological behavior of PTC cells.Methods The expressions of lncRNA FoxP4-AS1 in PTC cell lines (TPC-1, K1 cells) and normal thyroid follicular epithelial cells (Nthy-ori3-1 cells) were determined by qRT-PCR. In TPC-1 and K1 cells after transfection with FoxP4-AS1 overexpressing lentiviral vector or empty virus vector (negative control), the changes in biological functions were analyzed by CCK-8 assay, colony formation assay, EdU incorporation assay, Transwell assay and flow cytometry, respectively. The potential target genes of FoxP4-AS1 were predicted by GEPIA database and LncTar website, and then verified by Western blot analysis. Using above transfected cells, subcutaneous tumor models in mice were created, then, the growth of the xenograft tumors was observed and Ki-67 expressions in the tumors were determined by immunohistochemical staining. A preliminary analysis of the signaling pathways potentially associated with FoxP4-AS1 was performed by using the KEGG database.Results qRT-PCR results showed that the expression levels of lncRNA FoxP4-AS1 in the two types of PTC cells were significantly lower than that in the normal thyroid follicular epithelial cells (both P<0.05). Cell function experiments showed that the proliferation activity as well as invasion and migration abilities were significantly decreased with G₀/G₁ phase arrest in TPC-1 and K1 cells after transfection with FoxP4-AS1 overexpressing lentivirus vector compared with the negative control cells (all P<0.01). The GEPIA database and LncTar website predicted that there were potential interaction targets between FoxP4-AS1 and CDK4. Western blot results showed that the expression levels of CDK4/cyclinD1 in the two types of cells transfected with FoxP4-AS1 overexpressing lentivirus vector were significantly lower than those in their corresponding negative control cells (all P<0.05). In the subcutaneous tumor transplantation models, the PTC cells transfected with FoxP4-AS1 overexpressing lentivirus vector showed a slow growth in the mice, small volume of the formed tumor and low Ki-67 expression in the tumor compared with PTC cells transfected with empty virus vector. KEGG pathway enrichment analysis showed that the FoxP4-AS1 low expression was mainly enriched in PI3K-AKT-MTOR pathway, cell cycle, apoptosis, and immune regulation interactions, and the FoxP4-AS1 high expression was mainly enriched in DNA methylation and oxidative stress pathways.Conclusion The expression of lncRNA FoxP4-AS1 is down-regulated in PTC cells and is closely related to the malignant biological behavior of PTC cells. FoxP4-AS1 may exert its actions as a protective factor by suppressing the expression of CDK4/cyclinD1 and thereby inhibiting the proliferation of PTC cells.