Of 57 human Cytochrome P450 (CYP) enzymes, twelve metabolize 90% of xenobiotics. To our knowledge, no study has addressed the relation between enzyme dynamics and substrate promiscuity for more than three CYPs. Here, we show by constraint dilution simulations with the Constraint Network Analysis for the twelve isoforms that structural rigidity of the F/G-region is significantly inversely correlated to the enzymes’ substrate promiscuity. This highlights the functional importance of structural dynamics of the substrate tunnel.