Abstract. Afforestation on former pastures is widely promoted due to its potential to increase soil organic carbon sequestration while reducing CO2 emission. The establishment of a forest on a former pasture, however, might affect soil microbial community structure due to the alteration in substrate quality and thus impact carbon cycling in soils. To date, it still remains an open question if and how afforestation may alter the soil microbial community structure and related implications for soil organic matter stabilization. In addition, the majority of studies focuses on low altitude regions which results in uncertainties regarding the effects of afforestation on soil microbiology in mountainous regions. In this study, we aimed to investigate the consequences of afforestation of a subalpine pasture with Norway spruce (Picea abies L.) on the soil microbial community structure following 130 years of afforestation. We used a multi-proxy biomarker approach, including phospholipid fatty acids (PLFAs) and glycerol dialkyl glycerol tetraethers (GDGTs), to explore the shift in the microbial community structure following afforestation with increasing forest stand age. We found a significant increase in bacterial communities (Gram- and Gram+ bacteria) with increasing forest stand age compared to the pasture. This trend, however, was reversed with increasing forest age when considering GDGT biomarkers. We thereby conclude that the microbial community in the pasture and forests of different forest stand ages utilize different carbon substrates as food resource, which is a direct consequence of the modification in litter input after the conversion of a pasture to forests. Our data further suggests that an increase in the soil organic matter decomposition results from the alteration in the microbial community structure, which is especially evident in the subsoil of the 130-year-old forest stand ages.