Understanding factors affecting cell invasion influences the design of engineered constructs for tissue regeneration. The objective of this work was to investigate the effect of matrix stiffness on invasion of tumor cells through a synthetic hydrogel with well-defined properties. A novel star acrylate-functionalized polyethylene glycol-co-lactide (SPELA) macromer was synthesized to produce hydrogels with well-defined water content, elastic modulus, degree of crosslinking and hydrophilicity. The hydrogel was formed by photo-polymerization of the macromer with or without integrin-binding cell adhesive RGD peptide. Cell invasion experiments were carried out in a transwell with SPELA hydrogel as the invading matrix and 4T1 mouse breast cancer cells. The invading cells on the lower membrane side were counted with an inverted fluorescent microscope. The concentration of SPELA macromer ranged from 10-25 wt% and that of RGD ranged from 1x10-4 to 1x10-2 M. The shear modulus of the hydrogel varied from 200 Pa to 25 kPa as the SPELA concentration increased from 10 to 25 wt%. Cell invasion slightly increased with increasing RGD concentration. However, RGD concentration >1% resulted in a significant decrease in cell migration. As the matrix stiffness increased from 0.15 to 0.4, 3, 5, 6, 14, and 25 kPa the invasion rate decreased from 18.0 to 5.5, 6, 5.7, 5.2, 1.5, and 1.0 cells/mm2/h, respectively. There was a sharp decrease in invasion rate for matrix stiffness greater than 10 kPa. Results demonstrate that matrix stiffness plays a major role in invasion of tumor cell through a gelatinous matrix.