Litter-derived dissolved organic carbon (DOC) is considered to be a major source of stabilised C in soil. Here we investigated the microbial utilisation of litter-derived DOC within an entire soil profile using a stable isotope labelling experiment in a temperate beech forest. The natural litter layer of a Dystric Cambisol was replaced by ¹³C enriched litter within three areas of each 6.57 m⁻² for 22 months and then replaced again by natural litter (switching-off the ¹³C input). Samples were taken continuously from 0 to 180 cm depths directly after the replacement of the labelled litter, and 6 and 18 months thereafter. We followed the pulse of ¹³C derived from aboveground litter into soil microorganisms through depth and over time by analysing ¹³C incorporation into microbial biomass and phospholipid fatty acids. Throughout the sampling period, most of the litter-derived microbial C was found in the top cm of the profile and only minor quantities were translocated to deeper soil. The microbial ¹³C stocks below 30 cm soil depth at the different samplings accounted constantly for only 6–12% of the respective microbial ¹³C stocks of the entire profile. The peak in proportional enrichment of ¹³C in subsoil microorganisms moved from upper (≤ 80 cm soil depth) to lower subsoil (80–160 cm soil depth) within a period of 6 months after switch-off, and nearly disappeared in microbial biomass after 18 months (< 1%), indicating little long-term utilisation of litter-derived C by subsoil microorganisms. Among the different microbial groups, a higher maximum proportion of litter-derived C was found in fungi (up to 6%) than in bacteria (2%), indicating greater fungal than bacterial dependency on litter-derived C in subsoil. However, in contrast to topsoil, fungi in subsoil had only a temporarily restricted increase in litter C incorporation, while in the Gram-positive bacteria, the C incorporation in subsoil raised moderately over time increasingly contributing to the group-specific C stock of the entire profile (up to 9%). Overall, this study demonstrated that microorganisms in topsoil of a Dystric Cambisol process most of the recently deposited aboveground litter C, while microbial litter-derived C assimilation in subsoil is low.