Circulation and flushing inside the lagoon system of Guana Tolomato Matanzas National Estuarine Research Reserve (GTMNERR or GTM) have been studied using a three-dimensional numerical circulation model CH3D. The lagoon system includes two tidal inlets (St. Augustine Inlet and Matanzas Inlet). To ensure accuracy in model results, we extend the model domain to include a large portion of the coastal water and the Ponce de Leon Inlet in the south. Water levels on the open boundaries are provided by the East Coast (2001) ADCIRC Tidal Database. Model simulations of barotropic and baroclinic circulation from April 1 to May 31, 2004, produced reasonable water levels at numerous stations inside the GTM. Simulated salinity results are not as good because of the lack of freshwater inflow data inside the GTM and salinity data offshore. Using the simulated flow fields, we solve the three-dimensional transport equations for conservative species, and determine the flushing characteristics inside the GTM in terms of the 50% renewal time of the conservative species within each of eight segments, which are selected by considering the geographical features and proximity to tidal inlets and rivers. The flushing results indicate that tide is the most dominant flushing mechanism, while river and salinity are important flushing mechanisms for segments that are far from the tidal inlets. The normalized flushing times, defined as the 50% renewal time divided by the volume of the segment, are calculated for the eight segments and compared with each other. Comparing the “normalized” flushing time at all segments, a relative flushing ranking (RFR) is generated that ranks the “normalized” flushing time from the shortest to the longest as follows: segment 2 (includes St. Sebastian), segment 8 (near Ponce Inlet), segment 3 (includes Fort Matanzas), segment 7 (near Ponce Inlet), segment 1 (includes Pine Island), segment 4 (includes Pellicer Creek), segment 6 (includes High Bridge Road), and segment 5 (includes Bings Landing). This quantitative ranking of flushing characteristics inside the GTM is made possible because of the use of a three-dimensional numerical circulation and transport model that incorporates the effect of hydrodynamics on flushing. These results provide much more quantitative information than the simple empirical residence time indices (1–4) developed for the GTM in a previous study. CH3D was also applied to simulate the circulation during January 25 to February 3, 2006. Simulated currents inside the St. Augustine Inlet on February 2, 2006, compare favorably with the currents measured by Acoustic Doppler Current Profiler (ADCP). Model simulated flow rates through St. Augustine and Matanzas inlets reproduced measured data on July 1 and June 16, 2004, respectively. Model-simulated currents and water levels improved when the flooding-and-drying version of the model was used. The three-dimensional modeling approach can be used to provide sguidance on resource management and the development of sampling strategies for several ongoing and prospective biogeochemical studies in the GTM.