Introduction: Nitroglycerin is widely administered intravenously. The ability of Nitroglycerin to inhibit blood coagulation by inhibiting platelet activation is well established. Previously we demonstrated that nitroglycerin could also significantly increase partial thromboplastin times. Objective: The primary objective was to study whether the mechanism was by direct inhibition of thrombin by nitroglycerin. Method: Time-to-gel measurements were conducted in an in vitro assay where fibrinogen solutions were converted to fibrin gels at thrombin and fibrinogen concentrations within normal physiologic ranges. Results: Nitroglycerin directly inhibited thrombin from catalyzing fibrinogen gelation in a concentration-dependent manner that was statistically significant at nitroglycerin concentrations used for intravenous infusions. Conclusion: The nitroglycerin concentration-dependence of the delay in time-to-gel was quantitatively consistent with theoretical predictions of the concentration-dependence of inhibitors on enzymes following Michaelis-Menten kinetics.