Lime application and understory removal are two silvicultural practices commonly used to improve stand productivity in subtropical plantations. However, little is known about how the two practices, particularly in their interaction, affect soil respiration ( R_s). This knowledge gap limits our accurate assessment for the carbon balance in plantations under lime application and understory removal, which are usually applied simultaneously in practices. Here, using a field manipulation experiment, we assessed the effects of lime application, understory removal, and their interactions on R_s, soil heterotrophic respiration ( R_h), and soil autotrophic respiration ( R_a) in subtropical Eucalyptus plantations in southern China. We also examined the changes in soil pH, soil microbial communities (PLFAs), Eucalyptus fine root biomass after lime application and understory removal. Lime application inhibited R_s due to the decreased R_a and the contribution of understory root respiration ( R_U) to R_s. Understory removal also reduced R_s due to the decreased soil microbial and fungal PLFAs, and the associated R_h, as well as the R_U. Lime application decreased R_s by 16.48% in the presence of understory and only 4.49% when understory was removed. These results demonstrate that lime application and understory removal interact to affect R_s and highlight that understory vegetation can highly mediate the effect of lime application on R_s in subtropical Eucalyptus plantations in southern China.