We investigated the effect of substrate binding on the mechanical stability of mouse dihydrofolate reductase using single-molecule force spectroscopy by atomic force microscopy. We find that under mechanical forces dihydrofolate reductase unfolds via a metastable intermediate with lifetimes on the millisecond timescale. Based on the measured length increase of approximately 22 nm we suggest a structure for this intermediate with intact substrate binding sites. In the presence of the substrate analog methotrexate and the cofactor NADPH lifetimes of this intermediate are increased by up to a factor of two. Comparing mechanical and thermodynamic stabilization effects of substrate binding suggests mechanical stability is dominated by local interactions within the protein structure. These experiments demonstrate that protein mechanics can be used to probe the substrate binding status of an enzyme.