Thrombin is a serine protease involved in the blood coagulation reaction, and it shows maximum enzymatic activity in the presence of Na+ It has been supposed that Na+ binding promotes conversion from the inactive form, with a collapsed primary substrate pocket (S1 pocket), to the active form, with a properly formed S1 pocket. However, the evidence supporting this activation mechanism was derived from the X-ray crystallographic structures solved under nonphysiological conditions and using thrombin mutants; thus, it still remains elusive whether the activation mechanism is actually attributed to Na+ binding. To address the problem, we employed all-atom molecular dynamics simulations for both active and inactive forms of thrombin in the presence and absence of Na+ binding and examined the effect of Na+ binding on S1-pocket formation. In contrast to the conventional supposition, we revealed that Na+ binding does not prevent S1-pocket collapse virtually, but rather, the bound Na+ can move to the S1 pocket, thus blocking substrate access directly. Additionally, it was clarified that Na+ binding does not promote S1-pocket formation. According to these insights, we concluded that Na+ binding is irrelevant to the interconversion between the inactive and active forms of thrombin.