Thrombin has been studied as a paradigmatic protein of Na+- activated allosteric enzymes. Earlier structural studies suggest that Na+-binding promotes the thrombin-substrate association reaction. However, it is still elusive because (1) the structural change, driven by Na+-binding, is as small as the thermal fluctuation, and (2) the bound Na+ is close to Asp189 in the primary substrate binding pocket (S1-pocket), possibly preventing substrate access via repulsive interaction. It still remains a matter of debate whether Na+-binding actually promotes the reaction. To solve this problem, we examined the effect of Na+ on the reaction by employing molecular dynamics (MD) simulations. By executing independent 210 MD simulations of apo and holo systems, we obtained 80 and 26 trajectories undergoing substrate access to S1-pocket, respectively. Interestingly, Na+-binding results in a 3-fold reduction of the substrate access. Furthermore, we examined works for the substrate access and release, and found that Na+-binding is disadvantageous for the presence of the substrate in the S1-pocket. These observations provide the insight that the bound Na+ is essentially a negative effecter in thrombin-substrate stereospecific complex formation. The insight rationalizes an enigmatic feature of thrombin, relatively low Na+-binding affinity. This is essential to reduce the disadvantage of Na+-binding in the substrate-binding.