[Abstract] Objective:To detect the expression of transcription factor FOXP4 (Forkhead box P4) in laryngeal squamous cell carcinoma (LSCC) tissues and cell lines, and to investigate its effects on the proliferation, migration, invasion, cell cycle, and apoptosis of LSCC TU177 cells in vitro as well as to explore its relationship with epithelial-mesenchymal transition (EMT) process. Methods: A total of 40 pairs of tumor tissues and adjacent tissues that resected from LSCC patients were collected from the biological specimen bank of the Forth Hospital of Hebei Medical University between 2013 and 2015. The expression of FOXP4 in LSCC tissues and corresponding adjacent tissues was detected by qPCR. qPCR and Western blotting were used to detect the FOXP4 expression level in human LSCC cell lines (AMC-HN-8, TU177, TU686, and TU212). Small interfering RNA (si-RNA) was used to knock down FOXP4 expression in TU177 cells. The effects of FOXP4 knockdown on the proliferation, migration, invasion, cell cycle and apoptosis of TU177 cells were measured by MTS assay, clone formation assay, Transwell chamber migration and invasion assay, and flow cytometry, respectively. The mRNA levels of EMT markers N-cadherin, β-catenin, Vimentin, Twist, Snail and zine finger E box binding homeobox 1 (ZEB1) after transfection of si-FOXP4 in TU177 cells were detected by qPCR. The changes of protein levels of N-cadherin, β-catenin, Vimentin and Twist after FOXP4 knockdown were measured by Western blotting. Results: The expression of FOXP4 in LSCC tissues was significantly higher than that in adjacent tissues (P<0.05), and it was related to the TNM stage of tumors and lymph node metastasis (all P<0.05). The expression of FOXP4 in LSCC cells was higher than that in the adjacent tissues (P<0.05 or P<0.01). The expression of FOXP4 in TU177 cells transfected with si-FOXP4 was significantly lower than that in the control group (P<0.01). Compared with the control group, knocking down FOXP4 could inhibit the proliferation, migration and invasion but promote the apoptosis of TU177 cells in vitro (all P<0.01), block the cell cycle at G0/G1 phase (P<0.01), and reduce cell replication in S phase (P<0.01); in addition, knocking down FOXP4 could reduce the mRNA levels of N-cadherin, β-catenin, Vimentin, Twist, Snail, ZEB1 (P<0.05 or P<0.01) and the protein levels of N-cadherin, β-catenin, Vimentin, Twist in TU177 cells. Conclusion: The high expression of FOXP4 may be related to the occurrence and development of LSCC. FOXP4 knockdown can inhibit the proliferation, migration and invasion of laryngeal cancer cells in vitro, block cell cycle at G0/G1 phase, promote apoptosis, and may participate in the EMT process.