The establishment of the Antarctic Circumpolar Current (ACC) is one of the most important events of the Cenozoic for both global oceanic circulation and climate. The onset of this major current hinges on the opening of two major oceanic passages, the Drake Passage and the Tasmanian gateways that connect Pacific, Atlantic and Indian oceans, allowing a modern-like thermohaline circulation. For decades, the ACC onset has been considered as the trigger of the Oligocene glaciation at 33.7 Ma, which marks the beginning of the modern icehouse climate. Today, this scenario is debated. The main obstacle to evaluate the ACC influence on the Oligocene glaciation remains the ill-constrained timing of the Drake Passage gateway opening. Here, we analyse the geochemical composition and Sr isotope ratio of dated planktonic and benthic foraminifera from two IODP and ODP legs in the Southern Atlantic and Pacific oceans (SAO and PO, respectively) to assess the variability of seawater masses' chemical composition through time and to better constrain the timing of the Drake Passage gateway opening along the Eocene-Oligocene interval. These results, based on seawater paleo temperature (Mg/Ca molar ratios), redox (Ce/Ce* anomaly) and provenance (87Sr/86Sr) proxies, highlight a gradual seawater mass mixing between the SAO and PO from 31 Ma to 26 Ma. Combined with a reconsideration of the fossil fish teeth Neodymium isotope records, these geochemical tracers evidencing the SAO-PO interconnection depicts the Drake Passage gateway opening and deepening during this 31–26 Ma interval and thus, the timing of the ACC onset. Hence, antecedence of the Oligocene glaciation onset (at 33.7 Ma) relative to the ACC onset (31–26 Ma) implies that the ACC did not trigger the Oligocene glaciation and that the role of atmospheric pCO2 should be further considered.