The development of effective strategies to maintain/increase soil C is hindered by the poor process-level understanding of the impact of management practices on C preservation, particularly at different soil depths. Based on the distinct biogeochemistry existing across a soil profile, a depth-dependent response of organic matter (OM) to soil amendments was hypothesized. To test this, we investigated the effect on OM preservation of lime addition to the topsoil and the subsoil of a volcanic soil classified as Andosol – the mineral soil with the largest organic C content worldwide. We incubated samples from each soil layer with (or without) lime addition, in the presence or absence of ¹³C- and ¹⁵N-labeled plant residue (simulating plant C inputs to the soil). The fate of OM in bulk soil and OM-mineral associations of microaggregates was measured using conventional chemical analyses and nano-scale secondary ion mass spectrometry, respectively. The results revealed contrasting geochemical properties existing within the soil profile, i.e. pH and amount of allophane increasing with soil depth. Functional complexity of OM also increased with depth, as revealed by an increase in spatial and molecular heterogeneity of OM, i.e. a larger proportion of microbial-derived OM with high spatial separation found in the subsoil. Lime addition caused OM destabilization as denoted by the increase in the amount of water-extractable C in both the topsoil and the subsoil (P < 0.05). In the topsoil, OM coverage of mineral surfaces decreased with liming from 49% to 30% whereas the opposite was observed in the subsoil, where it increased from 23% to 29%. Thus, liming caused the disruption of OM-mineral associations within microaggregates in the topsoil, but not in the subsoil. We infer that, at depth, the OM destabilization involved the mobilization of OM previously protected within macroaggregates. This pulse of labile C generated an advantageous environment for microbial OM mineralization in the subsoil, but this effect was diluted in the topsoil, richer in OM and where OM functional complexity is smaller. Depth-dependent soil geochemical properties and functional complexity determine differences in the effect of liming on the fate of soil OM in top vs. subsoils.