Stratification increases in tropical and subtropical oceans due to global warming is predicted to inhibit primary marine productivity. Analysis of 17-years of satellite chlorophyll (Chl) data demonstrates increased Chl concentrations in the southern Indian Ocean (IO) with known rising sea surface temperature (SST). This seemingly bring the fundamental assumption in the stratification control model into question. Herein, using the newly available temperature/salinity observations, we re-evaluated stratification in the southern IO by calculating the squared buoyancy frequency (N2) down to depths of 2,000 m. This avoids the limitation of previous quantification of stratification that has depended on SST or temperature difference between the surface and depths of 200 m. We found that the southern IO experienced basin-wide surface warming, 50100 m cooling around 10°S, and faster rates of subsurface warming in the subtropical IO gyre. Specifically, we demonstrate that around 10°S, meridional and zonal advections contribute to the temperature increase, while vertical diffusion and advection decrease the temperature. In the gyre, vertical advection drives the temperature increase, which offsets the meridional advection and vertical diffusion-induced cooling. These inhomogeneous trends in the vertical thermohaline structure induce subsurface instability, making the surface stratification more likely to be disturbed in the condition of increasing heat lossthus increasing the potential for upward entrainment of nutrients into the euphotic zone. Our results highlight the importance of a three-dimensional framework for examining stratification to assess future marine ecosystem responses to a changing climate.