The Aegean Sea (NE Mediterranean) represents a key area for climatic and oceanographic future projections, therefore it is crucial to define its environmental evolution during the Pliocene paleoclimatic analogue. The DSDP Site 378, located in the South Aegean Cretan Basin, provides an exceptional opportunity to investigate in detail the paleoceanographic conditions of the Early Pliocene and to examine the basin's paleoenvironmental evolution, through the study of calcareous nannofossil accumulation rates and hydroclimate proxies. The sequence has been dated in between ∼5.2–3.9 Ma, with the age of the first Zanclean marine sediments overlying the Messinian Primary Lower Gypsum demonstrating that the Cretan Basin was partially desiccated for at least ∼400 kyrs, as a result of being a shallow epicontinental area in the Late Miocene. A relatively sudden change to deep marine environment took place in the Early Pliocene linked to the accelerated rate of trench extension in the Cretan Basin. The multi-proxy study revealed intervals reflecting warm surface waters and low-oxygen conditions on the seafloor, associated with the orbitally driven presence of Early Pliocene rhythmic sapropelic layers. Between ∼5.2 and 4.6 Ma, the sapropelic layers were characterized by elevated abundance of Reticulofenestra spp. and Florisphaera profunda, depicting increased productivity throughout the photic zone. Both enhanced riverine inputs and weak presence of Levantine Intermediate Water (LIW) characterized this time interval. The alterations in the calcareous nannofossil patterns with increased Helicosphaera spp. and Umbilicosphaera jafari abundances, implying freshening and enhanced mid-photic productivity after ∼4.6 Ma, suggest strengthening of LIW intrusion combined with the full restoration of North-South Aegean marine gateways that led to the establishment of a hydrographic system similar to the modern Aegean Sea.