Earthworms are important drivers for the formation of soil structure and play a key role in soil organic matter (SOM) dynamics. Our previous long-term (126 weeks) laboratory experiment showed that carbon (C) loss declined through time in soil when litter was mixed and consumed by earthworms (Lumbricus rubellus). Eventually, the C loss was lower than in treatments where litter was mechanically mixed into soil with exclusion of earthworms. However, it is not clear if the solely physical manipulation of soil or biological activity of earthworms lead to different SOM quality, which would result in a distinction in C loss and consequently C sequestration. Thus, we differentiated between physical (mechanical mixing) and earthworm effects on SOM composition. Two types of soil were used in the experiment: clay and sand, and these were incubated with alder (Alnus glutinosa) and willow (Salix caprea) litter, respectively. The combination of soils and litter types corresponds to the natural combinations at the sampling sites. To explain underlying mechanisms of a lower C loss in the earthworm vs. mechanically mixed treatment, we separated SOM fractions in order to gain pools defined in the Rothamsted model. Chemical differences between initial litter and the active and slow pool of SOM obtained by fractionation were studied. No significant differences between the earthworm and mechanically mixed treatment were found in C, nitrogen (N), and phenol contents, composition of major chemical groups of litter studied by solid-state ¹³C NMR spectroscopy, and composition of aromatic components of SOM studied by analytical pyrolysis (Py GC/MS). This lack of differences in chemical composition suggests that greater SOM sequestration in the earthworm treatment is likely to be connected with physical protection of SOM inside cast aggregates rather than with chemical changes in SOM mediated by earthworms.