Abstract The effect of earthworms on humus composition and humic acid (HA) characteristics was studied in a rice–wheat rotation agroecosystem. Experimental plots in the rotation had five treatments, i.e., incorporation or mulching of maize residues with or without added earthworms and a control. Compared with the control, the application of maize residues to soil either alone or in combinations with earthworms strongly affected the humus composition and HA characteristics. In the presence of earthworms, the carbon ratio of HA to fulvic acid (FA), and the alkyl C/O-alkyl C and hydrophobic C/hydrophilic C ratios of HA were slightly higher, while the aliphatic C/aromatic C ratio of HA was slightly lower following maize residue incorporation than the same treatment without earthworms. In contrast, these ratios generally remained almost constant following maize residue mulching. Thus, earthworm activity following maize residue incorporation was more favorable for the HA formation, and was associated with a decrease in aliphaticity of HA, and an increase in its degree of humification and hydrophobicity.