Age-related intimal stiffening is associated with increased endothelium permeability, an initiating step in atherosclerosis. Notably, in addition to a bulk increase in matrix stiffness, the aged intima also exhibits increased spatial stiffness heterogeneity. We investigate the effect of heterogeneous matrix stiffness on endothelial cells. Methacrylated hyaluronic acid hydrogels are fabricated and photopatterned to create substrates with 50-and 100 μm squares containing soft and stiff matrix regions of 2.7 ± 0.7 and 10.3 ± 3.9 kPa. On the patterned matrices, endothelial cells integrate subcellular matrix stiffness cues at stiffness interfaces, and focal adhesions are increased in the cell body adhered to stiff matrix regions. Increased matrix stiffness heterogeneity disrupts cell–cell junctions in confluent endothelial monolayers. Together, this work indicates that the spatial presentation of matrix mechanical cues, in addition to bulk substrate compliance, play a role in governing endothelial single cell and monolayer behaviors.