Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are bacterial membrane lipids whose distribution in peatland soils serves as an important proxy for past climate changes due to strong linear correlations with temperature in modern environments. However, commonly used brGDGT-based temperature models are characterized by high uncertainty (ca. 4 °C) and these calibrations can show implausible correlations when applied at an ecosystem level. This lack of accuracy is often attributed to our limited understanding of the exact mechanisms behind the relationship between brGDGTs and temperature and the potential effect of temperature-independent factors on brGDGT distribution. Here, we examine the abundance and distribution of brGDGTs in a boreal peatland after four years of in-situ warming (+0, +2.25, +4.5, +6.75 and +9 °C). We observed that with warming, concentrations of total brGDGTs increased. Furthermore, we determined a shift in brGDGT distribution in the surface aerobic layers of the acrotelm (0–30 cm depth), whereas no detectable change was observed at deeper anaerobic depths (>40 cm), possibly due to limited microbial activity. The response of brGDGTs to warming was also reflected by a strong increase in the methylation index of 5-methyl brGDGTs (MBT′5Me), classically used as a temperature proxy. Further, the relationship between the MBT′5Me index and soil temperature differed between 0–10, 10–20 and 20–30 cm depth, highlighting depth-specific response of brGDGTs to warming, which should be considered in paleoenvironmental and paleoecological studies. As the bacterial community composition was generally unaltered, the rapid changes in brGDGT distribution argue for a physiological adaptation of the microorganisms producing these lipids. Finally, soil temperature and water table depth were better predictors of brGDGT concentration and distribution, highlighting the potential for these drivers to impact brGDGT-based proxies. To summarize, our results provide insights on the response of brGDGT source microorganisms to soil warming and underscore brGDGTs as viable temperature proxies for better understanding of climatic perturbation in peatlands.