The late Miocene Messinian salinity crisis (MSC; 5.97–5.33 Ma) transformed the Mediterranean basin into the youngest salt giant in Earth history. The paleoenvironment and the paleodepth of the Mediterranean basin during the terminal phase of the MSC (i.e., the Lago-Mare event), are still uncertain, primarily due to the contradicting presence of in-situ, fully marine vs brackish to freshwater fossils, with different palaeoecological affinity, found in the same rock record. This study uses the distribution of C37, C38 and C39 long-chain unsaturated methyl and ethyl n-ketones (alkenones), produced by haptophyte algae, in combination with micropaleontological and stable oxygen isotope data, to reconstruct the surface water temperatures and paleoenvironmental conditions in the Northern Mediterranean basin across the transition between the latest Miocene Lago-Mare phase to the re-establishment of marine conditions starting in Pliocene, at 5.33 Ma. The results suggest that the Mediterranean basin was already receiving influxes from the Atlantic Ocean before the end of the MSC, resulting in a progressive return to marine conditions proceeding from south to north, i.e., from the more distal to proximal areas. Finally, fully marine conditions were established at the base of the Pliocene (5.33 Ma). The study highlights the reliability of alkenone-based paleo proxies for environmental reconstruction, especially when body fossil records of primary producers are scarce or problematic.