We present a species-specific Mg/Ca-calcification temperature calibration for Globorotalia inflata from a suite of 38 core top samples from the South Atlantic (from 8°S to 49°S). G. inflata is a deep-dwelling planktonic foraminifer commonly occurring in subtropical to subpolar conditions, which qualifies it for reconstructions of the permanent thermocline. Apparent calcification depths and calcification temperatures were determined by comparing measured δ18O with equilibrium δ18O of calcite based on water column properties. Based on our core top samples, G. inflata apparent calcification depth is constant throughout the South Atlantic midlatitudes with a depth of 350–400 m within the permanent thermocline. The resulting Mg/Ca-calcification temperature calibration is Mg/Ca = 0.72 ± 0.045/0.042 exp (0.076 ± 0.006 calcification temperature) (r2 = 0.81) and covers the temperature range 3.1°C–16.5°C. We applied our Mg/Ca calibration to gravity core PS2495-3 from the Mid-Atlantic Ridge at ∼41°S to test its validity by reconstructing a low-resolution record covering the last two glacial-interglacial cycles. Our paleotemperature record reveals large changes in temperature for Terminations I and II, when permanent thermocline temperature increased by as much as 8°C. The G. inflata paleotemperature record suggests that oceanic fronts repeatedly migrated over the location of core PS2495-3 during the last 160 kyr. This study shows the potential of G. inflata Mg/Ca to reconstruct paleotemperatures in the permanent thermocline.