Laryngeal squamous cell carcinoma (LSCC) is a common malignant tumor with a poor prognosis. Fascin actin-bundling protein 1 (FSCN1) has been reported to play a crucial role in the development and progression of LSCC; however, the underlying molecular mechanisms remain unknown. Herein, a whole transcriptome microarray analysis was performed to screen for differentially expressed genes (DEGs) in cells in which FSCN1 was knocked down. A total of 462 up and 601 downregulated mRNA transcripts were identified. Functional annotation analysis revealed that these DEGs were involved in multiple biological functions, such as transcriptional regulation, response to radiation, focal adhesion, extracellular matrix-receptor interaction, steroid biosynthesis and others. Through co-expression and protein-protein interaction analysis, FSCN1 was linked to novel functions, including defense response to virus and steroid biosynthesis. Furthermore, crosstalk analysis with FSCN1-interacting proteins revealed seven DEGs, identified as FSCN1-interacting partners, in LSCC cells, three of which were selected for further validation. Co-immunoprecipitation validation confirmed that FSCN1 interacted with prostaglandin reductase 1 and 24-dehydrocholesterol reductase (DHCR24). Of note, DHCR24 is a key enzyme involved in cholesterol biosynthesis, and its overexpression promotes the proliferation and migration of LSCC cells. These findings suggest that DHCR24 is a novel molecule associated with FSCN1 in LSCC, and that the FSCN1-DHCR24 interaction may promote LSCC progression by regulating cholesterol metabolism-related signaling pathways.