Extracellular matrix (ECM) proteins play an important role in the pathological processes of tumor development and progression. Elastic microfibril interface located protein-1 (EMILIN-1), an ECM glycoprotein, is linked to cell adhesion and migration. It was identified from head and neck squamous cell carcinoma (HNSCC) tissues that down-regulated EMILIN-1. It is associated with an increased risk of secondary primary malignancy development in HNSCC and hypothesized to function as a tumor suppressor in HNSCC. This study showed that EMILIN-1 expression in HNSCC tissues was specific to the stromal area, and secreted-EMILIN-1 level was higher in fibroblasts isolated from HNSCC tissues than in HNSCC cells. EMILIN-1 overexpression decreased cell proliferation, migration, and invasion in FaDu and CAL27 cells. Knockdown of EMILIN-1 in HNSCC cancer-associated fibroblasts induced cell proliferation and migration. The conditioned medium from EMILIN-1 knockdown cancer-associated fibroblasts increased HNSCC cell proliferation, and the co-culture system enhanced cancer cell migration and invasion. RNA-sequencing analysis revealed that the cell cycle and aurora kinase signaling were the most significant enrichment pathways, confirmed at the protein level. Furthermore, in an in ovo chick chorioallantoic membrane model, overexpression of EMILIN-1 in FaDu cells reduced tumor size and Ki-67-positivity and increased cleaved caspase-3-positive cells. These findings suggest that EMILIN-1 suppresses HNSCC growth partly through the down-regulation of cell cycle and aurora kinase signaling pathways.