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Despite chronic fibrosis occurring in many pathological conditions, few in vitro studies examine how fibrosis impacts lymphatic endothelial cell (LEC) behavior. This study examined stiffening profiles of PhotoCol®—commercially available methacrylated type I collagen—photo-crosslinked with the photoinitiators: Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), Irgacure 2959 (IRG), and Ruthenium/Sodium Persulfate (Ru/SPS) prior to evaluating PhotoCol® permeability and LEC response to PhotoCol® at stiffnesses representing normal and fibrotic tissues. Ru/SPS produced the highest stiffness (~6 kilopascal (kPa)) for photo-crosslinked PhotoCol®, but stiffness did not change with burst light exposures (30 and 90 s). The collagen fibril area fraction increased, and dextran permeability (40 kilodalton (kDa)) decreased with photo-crosslinking, showing the impact of photo-crosslinking on microstructure and molecular transport. Human dermal LECs on softer, uncrosslinked PhotoCol® (~0.5 kPa) appeared smaller with less prominent vascular endothelial (VE)-cadherin (cell–cell junction) expression compared to LECs on stiffer PhotoCol® (~6 kPa), which had increased cell size, border irregularity, and VE-cadherin thickness (junction zippering) that is consistent with LEC morphology in fibrotic tissues. Our quantitative morphological analysis demonstrates our ability to produce LECs with a fibrotic phenotype, and the overall study shows that PhotoCol® with Ru/SPS provides the necessary physical properties to systematically study LEC responses related to capillary growth and function under fibrotic conditions.