Millennial-scale Atlantic meridional overturning circulation (AMOC) variability has often been invoked to explain the Dansgaard–Oeschger (DO) events. However, the underlying causes responsible for millennial-scale AMOC variability are still debated. High-resolution U37K′ and TEX86H temperature records for the last 50 kyr obtained from the tropical Northeast (NE) Atlantic (core GeoB7926-2, 20°13′N, 18°27′W, 2500 m water depth) show that distinctive DO-type subsurface (i.e. below the mixed layer: >20 m water depth) temperature oscillations occurred with amplitudes of up to 8 °C in the tropical NE Atlantic during Marine Isotope Stage 3 (MIS3). Statistical analyses reveal a positive relationship between the reconstructed substantial cooling of subsurface waters and prominent surface warming over Greenland during DO interstadials. General circulation model (GCM) simulations without external freshwater forcing, the mechanism often invoked in explaining DO events, demonstrate similar anti-phase correlations between AMOC and pronounced NE Atlantic subsurface temperatures under glacial climate conditions. Together with our paleoproxy dataset, this suggests that the vertical temperature structure and associated changes in AMOC were key elements governing DO events during the last glacial.