Abstract: Glycerol dialkyl glycerol tetraethers (GDGTs) derived from microorganisms are important tools for the study of paloclimate changes. Incubation experiments are helpful to clarify the mechanisms for the responses of GDGTs to environmental parameters, and to test the reliability of related climatic proxies. However, previous GDGT incubation experiments were mainly conducted on a single strain or suffered from the influence of background signal, hampering systematically understanding the precise response of this biomarker to environmental factors in soil environment. In this study, artificial soils without GDGTs were incubated under the same temperature but different soil water content (SWC) conditions. The results showed that: (1) the abundances of GDGTs were positively correlated with SWC, but phosphate buffer could inhibit the production of GDGTs; (2) The branched and isoprenoid tetraether index (BIT), a soil moisture proxy developed in natural soils, was not significantly correlated with SWC; (3) 6-methyl brGDGTs were more abundant than 5-methyl brGDGTs, resulting in extremely high values of MBT'_5ME and low MBT'. Our results suggest that the BIT soil moisture proxy may indirectly (rather than directly) respond to SWC changes, and confirm that high relative abundance of 6-methyl brGDGTs can affect the applicability of the MBT'_5ME paleothermometer in soils. The BRIEF REPORT is available for this paper at http://www.ykcs.ac.cn/en/article/doi/10.15898/j.ykcs.202405240120.