Atomistic simulations of dioxygen (O2) dynamics and migration in nitric oxide-bound truncated Hemoglobin N (trHbN) of Mycobacterium tuberculosis are reported. From more than 100 ns of simulations the connectivity network involving the metastable states for localization of the O2 ligand is built and analyzed. It is found that channel I is the primary entrance point for O2 whereas channel II is predominantly an exit path although access to the protein active site is also possible. For O2 a new site compared to nitric oxide, from which reaction with the heme group can occur, was found. As this site is close to the heme iron, it could play an important role in the dioxygenation mechanism as O2 can remain there for hundreds of picoseconds after which it can eventually leave the protein, while NO is localized in Xe2. The present study supports recent experimental work which proposed that O2 docks in alternative pockets than Xe close to the reactive site. Similar to other proteins, a phenylalanine residue (Phe62) plays the role of a gate along the access route in channel I. The most highly connected site is the Xe3 pocket which is a “hub” and free energy barriers between the different metastable states are ≈1.5 kcal mol−1 which allows facile O2 migration within the protein.