Stable isotope data from eastern equatorial Pacific (EEP) core TR163-19 (2°15′N, 90°57′W, 2348 m) are presented for the surface-dwelling foraminifers Globigerinoides ruber and G. sacculifer and thermocline-dwelling Globorotalia menardii and Neogloboquadrina dutertrei. Using species-specific normalization factors derived from experimental and plankton tow data, we reconstruct a 360 kyr record of water column hydrography across the past three glacial cycles. We demonstrate that G. ruber maintains a mixed layer habitat throughout the entire record, while G. sacculifer records a mixture of thermocline and mixed layer conditions and G. menardii and N. dutertrei record thermocline properties. We conclude that G. sacculifer is not appropriate for paleoceanographic applications in regions with steep vertical hydrographic gradients. Results suggest that this region of the EEP had a thicker mixed layer and deeper δ13CDIC boundary between the surface and equatorial undercurrent during the last two glacial periods. A shift in N. dutertrei and G. sacculifer geochemistry prior to ∼185 kyr suggests water column structure and chemocline gradients changed, possibly due to a shift in the position of the undercurrent relative to this site. The timing and magnitude of glacial-interglacial δ13C variations between species indicates that near-surface carbon chemistry is controlled by changes in productivity, atmospheric circulation, and advected intermediate water sources north of the Antarctic polar front. These results demonstrate that when properly calibrated for species differences, multispecies geochemical data sets can be invaluable for reconstructing water column structure and properties in the past.