Tobacco smoke contains various cancer-causing toxic substances, including nicotine and nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). The cytochrome 2A13 is involved in nicotine metabolism and in the activation of the pro-carcinogenic agents NNK and NNN, by means of α-hydroxylation reactions. Despite the significance of cytochrome 2A13 in the biotransformation of these molecules, its conformational mechanism and the molecular basis involved in the process are not fully understood. In this study, we used molecular dynamics and principal component analysis simulations for an in-depth analysis of the essential protein motions involved in the interaction of cytochrome 2A13 with its substrates. We also evaluated the interaction of these substrates with the amino acid residues in the binding pocket of cytochrome 2A13. Furthermore, we quantified the nature of these chemical interactions from free energy calculations using the Molecular Mechanics/Generalized Born Surface Area method. The ligands remained favorably oriented toward compound I (cytochrome P450 O═FeIV state), to undergo α-hydroxylation. The hydrogen bond with asparagine 297 was essential to maintaining the substrates in a favorable catalytic orientation. The plot of first principal motion vs second principal motion revealed that the enzyme's interaction with nicotine and NNK involved different conformational subgroups, whereas the conformational subgroups in the interaction with NNN are more similar. These results provide new mechanistic insights into the mode of interaction of the substrates with the active site of cytochrome 2A13, in the presence of compound I, which is essential for α-hydroxylation.