Research on the development of collagen constructs is extremely important in the field of tissue engineering. Collagen scaffolds for numerous tissue engineering applications are frequently crosslinked with 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide hydrochloride (EDC) in the presence of N-hydroxy-succinimide (NHS). Despite producing scaffolds with good biocompatibility and low cellular toxicity the influence of EDC/NHS crosslinking on the cell interactive properties of collagen has been overlooked. Here we have extensively studied the interaction of model cell lines with collagen I-based materials after crosslinking with different ratios of EDC in relation to the number of carboxylic acid residues on collagen. Divalent cation-dependent cell adhesion, via integrins α$\mathrm{<mrow\; data-mjx-texclass="ORD">1</mrow>}$β$\mathrm{<mrow\; data-mjx-texclass="ORD">1</mrow>}$, α$\mathrm{<mrow\; data-mjx-texclass="ORD">2</mrow>}$β$\mathrm{<mrow\; data-mjx-texclass="ORD">1</mrow>}$, α$\mathrm{<mrow\; data-mjx-texclass="ORD">10</mrow>}$β$\mathrm{<mrow\; data-mjx-texclass="ORD">1</mrow>}$ and α$\mathrm{<mrow\; data-mjx-texclass="ORD">11</mrow>}$β$\mathrm{<mrow\; data-mjx-texclass="ORD">1</mrow>}$, were sensitive to EDC crosslinking. With increasing EDC concentration, this was replaced with cation-independent adhesion. These results were replicated using purified recombinant I domains derived from integrin α$\mathrm{<mrow\; data-mjx-texclass="ORD">1</mrow>}$ and α$\mathrm{<mrow\; data-mjx-texclass="ORD">2</mrow>}$ subunits. Integrin α$\mathrm{<mrow\; data-mjx-texclass="ORD">2</mrow>}$β$\mathrm{<mrow\; data-mjx-texclass="ORD">1</mrow>}$-mediated cell spreading, apoptosis and proliferation were all heavily influenced by EDC crosslinking of collagen. Data from this rigorous study provides an exciting new insight that EDC/NHS crosslinking is utilising the same carboxylic side chain chemistry that is vital for native-like integrin-mediated cell interactions. Due to the ubiquitous usage of EDC/NHS crosslinked collagen for biomaterials fabrication this data is essential to have a full understanding in order to ensure optimized collagen-based material performance.