Objective：This study aims to explore the influence and mechanism of mechanical properties of extracellular matrix on biological behaviors of oral squamous carcinoma cells. Methods：Two kinds of oral squamous carcinoma cells，CAL27 and HSC3，were cultured on hydrogel with different mechanical properties. Cell morphological changes were observed by microscope. The location and expression of laminin β2（LAMB2），collagen VI α1（COL6A1） and E-cadherin（CDH1） were detected by immunofluorescence. The mRNA expression level of SOX2，NANOG，LAMB2，COL6A1，integrin α6β4（ITGA6 and ITGB4） and CDH1 were detected by qPCR. Results：The mechanical properties of hydrogel changed the adhesion of oral squamous carcinoma cells，and the high mechanical properties of hydrogel increased cell adhesion. The biological behavior of cells on hydrogel with high mechanical properties is similar to those on traditional cell culture plates. CAL27 formed tight multicellular tumor spheroid on hydrogel with low mechanical properties，while HSC3 formed loose cell clusters. On hydrogels with low mechanical properties，the expression of LAMB2，COL6A1，integrin α6β4 in the two kinds of cells were increase. Cell stemness markers（SOX2，NANOG）and E-cadherin（CDH1）in CAL27 decreased with mechanical properties increased in hydrogels，whereas HSC3 were opposite. Conclusion：The mechanical properties of extracellular matrix can regulate cell adhesion，extracellular matrix protein synthesis，tumor cell stemness and intercellular adhesion. The mechanical properties of extracellular matrix may change the biological behavior of cells by changing the protein expression of extracellular matrix. However，the specific cell biological behavior is related to the specificity of cell line.