Nitric oxide (NO) is an important signaling molecule produced by a family of enzymes called nitric oxide synthases (NOS). Because NO is involved in various pathological conditions, the development of potent and isoform-selective NOS inhibitors is an important challenge. In the present study, the dimer of oxygenase domain of human iNOS (iNOSoxy) complexed to its natural substrate L-arginine (L-Arg) and both heme and tetrahydro-L-biopterin (BH4) cofactors was studied through multiple molecular dynamics simulations. Starting from the X-ray structure available for that complex (PDB: 1NSI), a 16 ns equilibration trajectory was first obtained. Twelve dynamics of slow extraction of L-Arg out from the iNOSoxy active site were then performed. The steered molecular dynamics (SMD) approach was used starting fromthree different points of the reference trajectory for a total simulation time of 35 ns. A probable unbinding/binding pathway of L-Arg was characterized. It was suggested that a driving force directed the substrate toward the heme pocket. Key intermediate steps/residues along the access route to the active site were identified along this "funnel shape" pathway and compared to existing data. A quasi-normal mode analysis performed on the SMDdata suggested that large collective motions of the protein may be involved in L-Arg binding and that opening the route to the active site in one monomer promoted an inverse, closing motion in the second monomer. Finally, our findings might help to rationalize the design of human iNOS isoform competitive inhibitors.