Computational modeling combined with mutational and activity assays was used to underline the substrate migration pathways in toluene 4-monooxygenase, a member of the important family of bacterial multicomponent monooxygenases (BMMs). In all structurally defined BMM hydroxylases, several hydrophobic cavities in the α-subunit map a preserved path from the protein surface to the diiron active site. Our results confirm the presence of two pathways by which different aromatic molecules can enter/escape the active site. While the substrate is observed to enter from both channels, the more hydrophilic product is withdrawn mainly from the shorter channel ending at residues D285 and E214. The long channel ends in the vicinity of S395, whose variants have been seen to affect activity and specificity. These mutational effects are clearly reproduced and rationalized by the in silico studies. Furthermore, the combined computational and experimental results highlight the importance of residue F269, which is located at the intersection of the two channels.