The Baltic Sea is a marginal sea sensitive to climate change. Relatively high sedimentation rates allow reconstructing Holocene climate variability with a high temporal resolution. The different TEX86 proxies (TEX86, TEX86L and TEX86H) based on Thaumarchaeota membrane lipids (isoprenoid glycerol dialkyl glycerol tetraethers; iGDGTs) represent a unique opportunity to estimate past water temperature variability as other temperature proxies are not applicable in the Baltic Sea. Although numerous TEX86 calibrations are available, the Baltic Sea represents a particular environment characterized by brackish and permanently stratified waters. Since surface sediments used to establish temperature calibrations are restricted in space and time, a downcore calibration approach is proposed here, which consists of comparing TEX86, TEX86L and TEX86H values from a well-dated sediment core with observed water temperatures in the central Baltic Sea over the last 45 years. The most reliable relationship is between TEX86L and water temperature at 80–120 m, the depth range in which Thaumarchaeota are thriving in the Baltic Sea. This result is corroborated by the absence of a seasonal pattern in the TEX86L index derived from a sediment trap time series. Comparing TEX86L water temperature estimates from a sediment core covering the last 160 years with hindcast simulations suggests that TEX86L estimates might be affected by changes in redox conditions. This is most likely related to a change in the depth habitat of Thaumarchaeota. The application of the new calibration on a long sediment core from the central Baltic Sea validates its efficiency to reconstruct subsurface water temperature in the Baltic Sea over the mid-to late Holocene.