The mammalian cytochrome P450 (CYP) enzymes play important roles in drug metabolism, steroid biosynthesis, and xenobiotic degradation. The active site of CYPs is buried in the protein and thus the ligands have to enter and exit the active site via ligand tunnels. Conformational changes of flexible parts of the protein usually accompany the entrance and exit of ligands. Comparison of the crystal structures of mammalian CYPs in closed, open, and partially open states reveals that the greatest conformational diversity associated with ligand tunnel opening is in the regions of the B–C and F–G loops. Some CYPs have been observed to adopt different open and closed conformations when bound to different ligands, suggesting that the ligand entrance and exit routes might differ according to the ligand properties. Mammalian CYPs are mostly membrane-bound enzymes, making them difficult to characterize structurally and dynamically. A range of molecular dynamics simulation techniques has been applied to investigate the dynamics and the ligand tunnels of these proteins both in the aqueous environment, and more recently, in lipid bilayers. These simulations not only reveal multiple tunnels through which ligands can pass but also show that different tunnels are preferred by different ligands and that the lipid bilayer can influence the protein dynamics and tunnel opening. The results indicate that not only the active site but also the ligand tunnels can contribute to the different substrate specificity profiles of the mammalian CYPs.