Significance The Cre- loxP system is a gene-editing tool that has enabled transformative advances in immunology, neuroscience, and cardiovascular research. Still, off-target activities confound research results and present obstacles to biomedical applications. Overcoming those limitations requires understanding the steps leading to assembly of recombination complexes, intasomes. We measured the magnetic properties of nitrogen nuclei in the backbone of the enzyme to correlate its intrinsic dynamics with its function in DNA recognition and cleavage. We found that in the absence of DNA the C terminus of Cre appears to block the DNA binding surface and active site of the enzyme. Binding to loxP DNA induces a conformational switch that would enable the intermolecular protein–protein interactions required for assembly of recombinogenic Cre intasomes.