The final phase of the closure of the Panamanian Gateway and the intensification of Northern Hemisphere Glaciation (NHG) both occurred during the Late Pliocene. Glacial–interglacial (G–IG) variations in sea level might, therefore, have had a significant impact on the remaining connections between the East Pacific and the Caribbean. Here, we present combined foraminiferal Mg/Ca and δ 18O measurements from Ocean Drilling Program (ODP) Site 1241 from the East Pacific and ODP Site 999 from the Caribbean. The studied time interval covers the first three major G–IG Marine Isotope Stages (MIS 95–100, ∼2.5 Ma∼2.5 Ma) after the intensification of NHG. Analyses were performed on the planktonic foraminifera Neogloboquadrina dutertrei and Globigerinoides sacculifer, representing water mass properties in the thermocline and the mixed-layer, respectively. Changes in sea water temperature, relative salinity, and water column stratification strongly suggest that the Panamanian Gateway temporarily closed during glacial MIS 98 and 100, as a result of changes in ice volume equivalent to a drop in sea level of 60–90 m. Reconstructed sea surface temperatures (SST) from G. sacculifer show a glacial decrease of 2.5 °C at Site 1241, but increases of up to 3 °C at Site 999 during glacial MIS 98 and 100 suggesting that the Panamanian Gateway closed during these glacial periods. The Mg/Ca-temperatures of N. dutertrei remain relatively stable in the East Pacific, but do show a 3 °C warming in the Caribbean at the onset of these glacial periods suggesting that the closing of the gateway also changed the water column stratification. We infer that the glacial closure of the gateway allowed the Western Atlantic Warm Pool to extend into the southern Caribbean, increasing SST (G. sacculifer) and deepening the thermocline (N. dutertrei). Additionally, ice volume appears to have become large enough during MIS 100 to survive the relatively short lasting interglacial MIS 99 so that the gateway remained closed. Towards the end of MIS 98, during MIS 97 and into MIS 96 temperatures on both sides are mostly similar suggesting water masses exchanged again. Additionally, Caribbean variations in SST and δ18Owater follow a precession-like cyclicity rather than the obliquity-controlled variations characteristic of the East Pacific and many other tropical areas, suggesting that regional atmospheric processes related to the trade winds and the InterTropical Convergence Zone (ITCZ) had a dominant impact in the Caribbean.