A 3D density structure of the lithosphere and upper mantle beneath the eastern Mediterranean Sea (EMS) and its adjacent region was constructed based on gravity anomaly inversion constrained by a seismic tomography model. Gravity effects of terrain and crust were removed from the observed gravity field (EIGEN-6C4) to obtain the residual mantle gravity anomaly (RMGA). The density distribution of the lithosphere and upper mantle was investigated. The 3D inversion process was constrained by an initial density model projected from the shear-wave velocity model (SL2013sv). The results show some characteristics of the density distribution in the lithosphere and in the upper mantle that could be related to the tectonic importance of the Mediterranean Sea and the surrounding region. A low-density zone dominates the lithosphere beneath the Sea except for the area around the Arabia Shield and North Anatolian Fault belt. A thinner, high-density layer beneath the southwest of the Sea may be related to the older oceanic lithospheric fragments. The high-density anomalies appear at depths below 280 km beneath the Sea and the Turkish Aegean Sea Plate. However, the low-density anomalies appear on the upper mantle under the trenches of the southwestern part of the Mediterranean Sea, the eastern part of the Aegean Sea, the Red Sea, the Black Sea, and the middle of the Arabia shield. The deep structure under the Eratosthenes seamount in the Mediterranean Sea is the source of the intensity and genesis of tectonic activity. Furthermore, the convergence region of two low-density anomaly zones (Africa-Anatolia) may be interpreted as a significant tectonic unit (Eratosthenes seamount) caused by the arrival of the relatively thick and buoyant Eratosthenes block to its present location south of Cyprus in Holocene time based on the density model interpretation beneath the Mediterranean Sea during the Late Cretaceous and early Tertiary period.